Authors: Anil P. Bidkar (1Department of Radiology and Biomedical Imaging, University of California, San Francisco, San Francisco, California.), Robin Peter (1Department of Radiology and Biomedical Imaging, University of California, San Francisco, San Francisco, California.; 2Department of Nuclear Engineering, University of California, Berkeley, California.), Anju Wadhwa (1Department of Radiology and Biomedical Imaging, University of California, San Francisco, San Francisco, California.), Kondapa Naidu Bobba (1Department of Radiology and Biomedical Imaging, University of California, San Francisco, San Francisco, California.), Scott Bidlingmaier (3Department of Anesthesia, University of California, San Francisco, San Francisco, California.), Niranjan Meher (1Department of Radiology and Biomedical Imaging, University of California, San Francisco, San Francisco, California.; 4National Institute of Pharmaceutical Education and Research, Lucknow, India.), Jonathan Chou (5Division of Hematology/Oncology, Department of Medicine, University of California, San Francisco, San Francisco, California.; 6UCSF Helen Diller Family Comprehensive Cancer Center, San Francisco, California.), Nancy Greenland (7Department of Pathology, University of California, San Francisco, San Francisco, California.), Chandrashekhar Dasari (8Division of Vascular and Endovascular Surgery, University of California, San Francisco, San Francisco, California.), Shubhankar Naik (9University of California, Berkeley, California.), Athira Raveendran (1Department of Radiology and Biomedical Imaging, University of California, San Francisco, San Francisco, California.), Megha Basak (1Department of Radiology and Biomedical Imaging, University of California, San Francisco, San Francisco, California.), Juan Antonio Camara Serrano (6UCSF Helen Diller Family Comprehensive Cancer Center, San Francisco, California.), Veronica Steri (6UCSF Helen Diller Family Comprehensive Cancer Center, San Francisco, California.), Scott Kogan (10Department of Laboratory Medicine, University of California, San Francisco, San Francisco, California.), Adam Oskowitz (8Division of Vascular and Endovascular Surgery, University of California, San Francisco, San Francisco, California.), Jiang He (11Department of Radiology and Medical Imaging, University of Virginia, Charlottesville, Virginia.), David M. Wilson (1Department of Radiology and Biomedical Imaging, University of California, San Francisco, San Francisco, California.; 6UCSF Helen Diller Family Comprehensive Cancer Center, San Francisco, California.), Rahul Aggarwal (5Division of Hematology/Oncology, Department of Medicine, University of California, San Francisco, San Francisco, California.; 6UCSF Helen Diller Family Comprehensive Cancer Center, San Francisco, California.), Renuka Sriram (1Department of Radiology and Biomedical Imaging, University of California, San Francisco, San Francisco, California.), Henry F. VanBrocklin (1Department of Radiology and Biomedical Imaging, University of California, San Francisco, San Francisco, California.; 6UCSF Helen Diller Family Comprehensive Cancer Center, San Francisco, California.), Youngho Seo (1Department of Radiology and Biomedical Imaging, University of California, San Francisco, San Francisco, California.; 2Department of Nuclear Engineering, University of California, Berkeley, California.; 6UCSF Helen Diller Family Comprehensive Cancer Center, San Francisco, California.), Bin Liu (3Department of Anesthesia, University of California, San Francisco, San Francisco, California.; 6UCSF Helen Diller Family Comprehensive Cancer Center, San Francisco, California.), Robert R. Flavell (1Department of Radiology and Biomedical Imaging, University of California, San Francisco, San Francisco, California.; 6UCSF Helen Diller Family Comprehensive Cancer Center, San Francisco, California.; 12Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, California.)
Categories: Precision Medicine and Imaging
Source: Clinical Cancer Research
Effective Treatment of Disseminated Prostate Cancer Using CD46-Targeted ^225^Ac Therapy
Authors: Anil P. Bidkar, Robin Peter, Anju Wadhwa, Kondapa Naidu Bobba, Scott Bidlingmaier, Niranjan Meher, Jonathan Chou, Nancy Greenland, Chandrashekhar Dasari, Shubhankar Naik, Athira Raveendran, Megha Basak, Juan Antonio Camara Serrano, Veronica Steri, Scott Kogan, Adam Oskowitz, Jiang He, David M. Wilson, Rahul Aggarwal, Renuka Sriram, Henry F. VanBrocklin, Youngho Seo, Bin Liu, Robert R. Flavell
Abstract
Purpose:
Metastatic castration-resistant prostate cancer has limited treatment options and a poor prognosis. Recently, prostate-specific membrane antigen (PSMA)-targeted alpha-particle therapy agents using actinium-225 (^225^Ac) have shown promising results in prostate cancer treatment, but a significant fraction of patients with advanced mCRPC demonstrate loss of PSMA expression. We have previously reported that PSMA-null and PSMA-positive tumors can be detected and treated effectively with CD46-targeted radiopharmaceuticals. This study evaluates the CD46-targeting PET imaging agent [^89^Zr]DFO-YS5, and the radioimmunotherapy agent [^225^Ac]Macropa-PEG4-YS5, in disseminated prostate cancer tumors.
Experimental Design:
Microtumor lesions, primarily observed in the liver, kidneys, and lungs, were successfully detected with [^89^Zr]DFO-YS5 PET imaging. We used disseminated 22Rv1 tumors for biodistribution studies, dosimetry assessments, and therapeutic efficacy evaluations of [^225^Ac]Macropa-PEG4-YS5.
Results:
Quantitative digital alpha-particle autoradiography revealed high radiation dose deposition from [^225^Ac]Macropa-PEG4-YS5 in microtumors compared with surrounding liver tissues, although in larger lesions (>1 mm diameter), the dose distribution was heterogeneous. Early treatment of smaller disseminated tumors with a uniform radiation dose was more effective in ablating tumors and promoting survival. In late-stage lesions of large size, heterogeneous dose deposition limited therapeutic efficacy, requiring higher administered activity to achieve a complete response.
Conclusions:
Our findings highlight that [^225^Ac]Macropa-PEG4-YS5 holds the potential for clinical translation for metastatic prostate cancer and reinforces the value of microdosimetry in understanding the efficacy of and resistance to targeted alpha therapy.
See related commentary by Patel et al., p. 2847
Translational RelevanceProstate cancer tumors can be effectively targeted using CD46-specific theranostic agents. This report highlights the dosimetry and treatment of disseminated prostate cancer tumors. The combination of high linear energy transfer and the short-range effects of alpha-particle therapy with [^225^Ac]Macropa-PEG4-YS5 has shown successful treatment outcomes in early-stage disseminated disease.
Introduction
Prostate cancer, which may progress into lethal metastatic castration-resistant prostate cancer (mCRPC), is the most common noncutaneous malignancy in men (1). Despite various available treatments, including androgen deprivation therapy, chemotherapy, immunotherapy, and radiotherapy, achieving a durable response in mCRPC remains a challenge (2). Recent developments in radioligand therapy (RLT) for prostate cancer have shown promising outcomes, highlighted by the approval of the RLT agent ^177^Lu-PSMA-617 (Pluvicto) for advanced forms of prostate cancer (3). Simultaneously, encouraging results are being reported from investigations into the therapeutic potential of ^225^Ac-PSMA-617 for patients with mCRPC (4). In parallel to these advancements, active research on the prostate-specific membrane antigen (PSMA)-targeting antibody J591 labeled with alpha or beta particle–emitting isotopes is gaining traction. The targeted delivery of ^225^Ac-J591 and ^177^Lu-J591 has demonstrated promising preliminary results in reducing tumor burden and improving patient outcomes in mCRPC (5–7). These RLT options promise to improve outcomes for patients with metastatic prostate cancer who have become resistant to other treatments. However, although many patients have a therapeutic benefit from ^225^Ac-PSMA-617 and ^177^Lu-PSMA-617 treatment, the responses are often not durable, as evidenced by eventual patient relapse (8). Furthermore, PSMA-targeted radiotheranostic molecules face limitations in PSMA-negative patients, including those with treatment-emergent small-cell neuroendocrine prostate cancer, in which decreases in PSMA expression often develop after treatment (9–11). Therefore, there is an unmet clinical need for additional targets for prostate cancer RLT, and several are now under investigation, including DLL3 (12), hK2 (13), CDCP1 (14), GRPR (15), and others (16).
Analysis of publicly available datasets has indicated high and homogeneous expression of CD46 mRNA in both adenocarcinoma and neuroendocrine prostate cancer phenotypes (17). Using a nongene expression–based antibody library selection approach, we identified prostate cancer–specific antibodies and CD46 as a novel cell surface antigen expressed on both PSMA-negative and PSMA-positive tumors (17–20). We identified a fully human antibody (YS5) that specifically binds to a tumor-selective CD46 epitope with minimal binding to normal cells and developed an antibody–drug conjugate (ADC) that selectively kills prostate cancer in vitro and in vivo (17). We also found that CD46 is overexpressed in multiple myeloma, and the CD46 ADC eliminated myeloma cells in vivo (21). Our CD46 ADC is currently being tested in phase I trials for prostate cancer (NCT03575819; ref. 22) and multiple myeloma (NCT03650491). Additionally, our immunoPET imaging agent, ^89^Zr-DFO-YS5, has shown efficacy in detecting prostate and multiple myeloma lesions (23, 24). This immunoPET agent is currently undergoing clinical testing in patients with mCRPC (NCT05245006) and patients with multiple myeloma (NCT05892393). Recently, we have successfully formulated alpha-particle therapy agents utilizing the YS5 antibody, including ^225^Ac-DOTA-YS5 (25), ^212^Pb-TCMC-YS5 (26), and [^225^Ac]Macropa-PEG4-YS5 (27, 28). [^225^Ac]Macropa-PEG4-YS5 demonstrates particular promise for treatment, as it includes the highly efficient Macropa chelator, a short PEGylated linker, and a stable amide linkage to the protein, resulting in efficient conjugation chemistry, high tumor retention and tumor-to-background ratios, and rapid urinary excretion of undesired systemic activity.
Tumor relapse following RLT is attributed to metastatic growth and insufficient radiation doses to eliminate tumor cells completely. Dosimetry may be used to measure the absorbed radiation dose to tumors and healthy tissue following RLT. Dosimetry methods demonstrate promise for understanding the expected response from RLT (29, 30). Additionally, understanding the stages of metastatic progression in prostate cancer is crucial for refining treatment strategies to minimize relapse risk (31). Preclinical tumor models of prostate cancer play a pivotal role in advancing our understanding of the disease (32). Patient-derived subcutaneous xenograft models, replicating primary tumor characteristics, serve as convenient tools (25). Although metastatic tumor models offer an improved representation for testing agents in preclinical settings, developing preclinical metastatic tumor models is challenging, owing to heterogeneous tumor dissemination and practical challenges in monitoring tumor progression.
Alpha-particle therapy with actinium-225 (^225^Ac) demonstrates great promise for treating various malignancies (33). The high linear energy transfer and short range of alpha particles make them suitable candidates for treating micrometastatic tumors. Recently, alpha-particle therapy agents (^225^Ac-PSMA-617, ^225^Ac-DOTATATE, ^225^Ac-PSMA-I&T, and ^225^Ac-J591, among others) have been developed and are being evaluated for human use (4, 5, 34, 35). In this work, we report the development, microscale dosimetry, and alpha-particle therapy of disseminated models of prostate cancer. Bioluminescence imaging (BLI) and small-animal PET (µPET) with [^89^Zr]DFO-YS5 probe were utilized to track tumor growth. The alpha-particle therapy agent, [^225^Ac]Macropa-PEG4-YS5, was used to study the dosimetry, biodistribution, and therapeutic response in the 22Rv1-Luc disseminated tumor model. High-resolution quantitative digital autoradiography and microdosimetry estimates revealed high dose deposition in the tumors although it became heterogeneous and lower in larger tumors. Treatment with [^225^Ac]Macropa-PEG4-YS5 significantly inhibited the growth of the disseminated tumors, particularly at an early stage of the disease, in which complete response (CR; with 90% survival) was observed.
Materials and Methods
Cell culture
Prostate cancer cells 22Rv1 (RRID: CVCL_1045), DU145 (RRID: CVCL_0105), PC3 (RRID: CVCL_0035), C42B (RRID: CVCL_4784), and LNCaP (RRID: CVCL_0395) were obtained from ATCC. All the cells were cultured in a CO2 incubator with RPMI medium supplemented with 10% FBS and an antibiotic solution (1% penicillin and streptomycin). All the cells used in our study were checked for mycoplasma contamination using the MycoAlert Mycoplasma Detection Kit (tested on October 2, 2023). Cells were authenticated by short tandem repeat phenotyping from ATCC. Cells were used for intracardiac inoculation after two passages following thawing. In order to track the growth of tumors, prostate cancer cells were transduced with a lentivirus packaged with a luciferase (Luc) vector under Neo/G418 selection and driven by a cytomegalovirus promoter.
Antibody and conjugation
A recombinant human YS5 antibody was produced in mammalian cells (Expi-HEK293) and purified by protein A chromatography and ion-exchange chromatography, as described (17). ^89^Zr radiolabeling was performed according to the method described by Wang and colleagues (23). Macropa-PEG4-YS5 conjugation was performed as described (28).
^225^Ac radiolabeling
The alpha-particle therapy agent [^225^Ac]Macropa-PEG4-YS5 was prepared according to the previously reported protocol (28). ^225^Ac received from Oak Ridge National Laboratory was dissolved in 0.2 mol/L HCl. For radiolabeling, a small volume (1–2 µL, 1.1 MBq) was added to the tube containing 50 µL of 2 mol/L NH4OAc (pH 5.8) and 20 µL of L-ascorbic acid (0.85 mol/L). Following this, 120 µg of Macropa-PEG4-YS5 was added to the reaction tube. The radiolabeling was performed at 37°C with constant stirring for 30 minutes. The radiolabeled antibody was purified using 30 kDa molecular weight cutoff centrifuge filters. The purity of [^225^Ac]Macropa-PEG4-YS5, confirmed with instant thin-layer chromatography, was 99.5%, with an isolated yield of 0.85 MBq and a molar activity of 1.02 GBq/µmol.
Animal studies
Male NOD/SCID gamma (NSG) (strain #005557, The Jackson Laboratory) mice (age 5–6 weeks) were used in this study. The experimental conditions and protocols were approved by the Institutional Animal Care and Use Committee and the Laboratory Animal Resource Center, University of California, San Francisco.
Biodistribution studies of [^225^Ac]Macropa-PEG4-YS5
Biodistribution of [^225^Ac]Macropa-PEG4-YS5 was studied in 1- and 3-week-old metastatic tumors of 22Rv1-Luc. Briefly, a 9.2 kBq dose of [^225^Ac]Macropa-PEG4-YS5 was injected into the mice, and the organs were collected on day 4. The collected organs were counted on the Hidex automatic gamma counter using a window of 25 to 2,000 keV without secular equilibrium (28).
Magnetic beads–based binding assay
The binding fraction of [^225^Ac]Macropa-PEG4-YS5 was determined using our previously published protocol (25). Briefly, three sets of sample tubes (A, B, and C) were prepared in triplicate. Each tube was loaded with 50 µL of magnetic beads. The magnetic beads were washed with 0.1% PBS Tween, followed by blocking in 1% milk PBS. CD46 antigen (10 µg) was added to tubes A and B, whereas tube C served as a no-antigen control. Subsequently, tube B received a 100-fold excess of cold YS5 for blocking, and all tubes were incubated for 30 minutes. After the blocking step, [^225^Ac]Macropa-PEG4-YS5 (100 ng per tube) was added to each tube and incubated for 20 minutes. The tubes were then washed three times with washing buffer. The bound activity was measured immediately using a Hidex gamma counter.
^89^Zr radiolabeling
The DFO-conjugated YS5 was developed according to the method described in our earlier study (23). The radiolabeling reaction was performed with 200 µg of DFO-YS5, 2 mCi (5 µL) of ^89^Zr oxalate, and 5 µL of 1 mol/L Na2CO3. The radiolabeled antibody [^89^Zr]DFO-YS5 was purified with PD10 column purification. The radiolabeling yield was found to be 95%, with 99% purity of the purified product.
Biodistribution studies of [^89^Zr]DFO-YS5
Biodistribution of [^89^Zr]DFO-YS5 was studied 1 and 3 weeks after intracardiac inoculations of 22Rv1-Luc cells in NSG mice. The mice were injected with [^89^Zr]DFO-YS5 (3.7 MBq dose) along with native IgG (0.5 mg/kg), followed by PET imaging and BLI imaging on day 4. The animals were euthanized, and organs were collected and counted on the Hidex automatic gamma counter (23).
Cell-binding assay
22Rv1 and CD46 knockout (KO) cells were seeded in the six-well plates at a density of 1 million cells per well. A blocking control was kept where cells were incubated with a 10-fold excess of cold YS5 for 1 hour. Following this, the cells were treated with [^225^Ac]Macropa-PEG4-YS5 at a concentration of 7 nmol/L (14 kBq activity) for 1 hour. Subsequently, cells were washed with PBS, and uptake was calculated using a gamma counter.
Colony formation assay
22Rv1 and 22Rv1-CD46 KO cells were seeded in the six-well plate assay at a density of 100 cells per well. Cells were treated with (0.003–0.37 kBq/mL) activities of [^225^Ac]Macropa-PEG4-YS5 for 96 hours. Following this, cells were washed with PBS three times, and fresh medium was added to the wells. Cells were incubated for colony formation for 2 weeks. Colonies were fixed and stained with crystal violet dye (1% w/v in 4% formaldehyde in water). The percentage of colonies was calculated.
DNA damage analysis after treatment of [^225^Ac]Macropa-PEG4-YS5
22Rv1 cells were seeded in multichamber slides. The treatment of [^225^Ac]Macropa-PEG4-YS5 was given with a radioactivity dose of 0.003 to 0.37 kBq/mL. After 96 hours of treatment, cells were washed with PBS and fixed with 4% formaldehyde for 10 minutes. Fixed cells were permeabilized with 1% TBST, followed by blocking with 2% nonfat milk in PBS. The cells were subsequently stained with primary antibody for phospho-γ-H2AX (1:300 dilution, anti-γ H2AX, phospho S139 antibody, Abcam #ab81299, RRID: AB_1640564) and the secondary antibody [1:1,000 dilution, anti-rabbit IgG, F(ab′)2 Fragment, Alexa Fluor 647 conjugate, #4414, RRID: AB_10693544]. The nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI; 1,000 dilution from 1 mg/mL stock). The images were captured with a ZEISS LSM 780 confocal microscope. The γ-H2AX foci were quantified using the CellProfiler program, in which nuclei were first identified using DAPI fluorescence, and the H2AX foci within the nuclear regions were counted. Three images from each treatment, each containing at least six cell nuclei, were analyzed.
Disseminated tumor model development
Prostate cancer (22Rv1-Luc, PC3-Luc, C42B-Luc, DU145-Luc, and LNCaP-AR-Luc) disseminated models were prepared by intracardiac injections. Briefly, Luc-expressing prostate cancer cells (100,000 cells per mouse) were injected into the left ventricle of the mice (NOD.Cg-Prkdc^scid^Il2rg^tm1Wjl^/SzJ, strain #005557, RRID: BCBC_4611) under ultrasound guidance (Vevo2100, FUJIFILM VisualSonics; ref. 36). Following intracardiac injection, tumor growth was measured by BLI (IVIS50 Imaging System) once a week. BLI images were acquired for 60 seconds for each scan, and the BLI intensity of each mouse was calculated by drawing a region of interest using Living Image 4.0 software.
Digital autoradiography and dosimetry analysis
Quantitative activity images were acquired using a digital autoradiograph (iQID, QScint Imaging Solutions, LLC) and converted into dose-rate maps using a previously validated dose-kernel protocol for single slices (37). Spatial dose-rate maps were coregistered to the adjacent hematoxylin and eosin (H&E) slice images through automated rigid body (translation and rotation) intensity maximization followed by manual landmark-to-landmark affine (translation, rotation, and shear) transformation (ImageJ BigWarp). We segmented each H&E slice into normal liver and tumor regions to analyze the coregistered autoradiographs. Two-dimensional dose-rate autoradiographs and segmented H&E masks were further Z-stacked to generate 3D renders of the liver and microtumors (38). Single-cell dosimetry calculations were conducted in a selected microtumor. The number of nuclei in a cross-section was counted from the H&E image using watershed segmentation and ImageJ Analyze Particles.
Spheroid autoradiography, viability, and DNA damage study
To form 3D 22Rv1 spheroids, cells were seeded in the low attachment surface plates at a density of 20,000 cells per well. Plates were centrifuged at 500 × g for 5 minutes to form cell clusters. Following this, the plates were incubated for 7 days to form spheroids. Cell viability of spheroids was tested following the treatment of [^225^Ac]Macropa-PEG4-YS5 for 96 hours. Following the treatment for 96 hours, cells were incubated with CellTiter-Glo 3D for 25 minutes. Spheroid cell viability was determined by taking luminescence with a Tecan microplate reader. For digital autoradiography, spheroids were treated with radioactivity for 96 hours. Subsequently, spheroids were washed with PBS five times, followed by the acquisition of decay events in iQID digital autoradiography. Similarly, for the DNA damage assay, spheroids treated with [^225^Ac]Macropa-PEG4-YS5 were washed with PBS, fixed, and stained with primary antibody (anti-γ H2A.X, phospho S139 antibody, Abcam, #ab81299, RRID: AB_1640564) and the secondary antibody [anti-rabbit IgG, F(ab′)2 Fragment, Alexa Fluor 647 conjugate, #4414, RRID: AB_10693544]. The images were captured with a ZEISS LSM 780 confocal microscope.
BLI and micro positron emission tomography (µPET) imaging
BLI and µPET imaging combined with CT (µPET/CT) were performed according to our previously published protocol (20). For BLI, mice were administered 100 µL of luciferin solution (1 gm/33 mL in PBS) by intraperitoneal injection. Bioluminescence images were acquired with the IVIS50 Imaging System. For µPET/CT imaging (nanoScan PET/CT, Mediso) with [^89^Zr]DFO-YS5 radiotracer probe, mice were injected with 3.7 MBq purified activity of [^89^Zr]DFO-YS5 antibody, along with 0.5 mg of cold IgG (for Fc blocking).
Histology
Adjacent sections of 10 µm thickness were used for digital autoradiography, histology, and immunofluorescence (IF) analysis. For histologic staining of the tissues, the tissues were frozen in an optimum cutting temperature medium. Optimum cutting temperature blocks of the tissues were sectioned (10 µm) with Cryotome. Tissue slides were stained according to the manufacturer’s protocol using the H&E staining kit (Abcam, #ab245880). Photographs were taken on a Nikon Eclipse 80i microscope with a Nikon Digital Sight camera using NIS-Elements F4.30 software at a resolution of 2,560 × 1,920.
IF (phospho-γ-H2AX) staining of liver sections for DNA damage analysis
The tissue sections were processed in acetone for 10 minutes, followed by methanol for 10 minutes. Rehydration of the slices was performed in 70%, 50%, and 30% ethanol for 2 minutes, followed by PBS. Permeabilization was performed in 1% TBST in blocking buffer (4% nonfat dry milk). Staining was performed overnight with primary antibodies. The following day, after washing steps, secondary staining along with DAPI was performed. The primary antibodies used were anti-CD46 antibody (Abcam, #ab273583) and anti-γ H2AX (Abcam, #ab81299). Secondary antibodies used were anti-rabbit IgG (Alexa Fluor 488 conjugate, Abcam, #4412) and anti-rabbit IgG (Alexa Fluor 647 conjugate, Abcam, #4414). The images were processed with CellProfiler for DNA damage analysis. Initially, we identified and counted nuclei using DAPI, which stains the nucleus. Following this, fluorescence from H2AX foci within the nuclear region was used to quantify γ-H2AX foci. Foci counting was achieved by setting thresholds for pixel intensity, allowing differentiation between background staining and actual foci. This method enables the automated and unbiased quantification of DNA damage across multiple images. The cell layers surrounding the tumor, each 10 µm thick, were determined by calculating the average distance between adjacent nuclei. H2AX foci were counted in the cells surrounding the tumor tissue (first layer) and in neighboring cells (second layer).
Toxicity analysis
For toxicity assessment, blood and serum from mice in the early and late treatment groups were analyzed for complete blood count (CBC), liver and kidney function tests, and histology staining. Blood and serum samples were analyzed in the Comparative Pathology Laboratory at UC Davis School of Veterinary Medicine.
3D digital autoradiograpy rendering
Stacks of 2D dose-rate autoradiographs or segmented H&E images were centered and registered using only rigid-body transformations by matching landmark microtumors between slices (ImageJ BigWarp, RRID: SCR_003070). Minor misalignments that may have been introduced by sample stretching or spacing between slices did not affect results because this 3D registration was only used for visualization. Once registered, images were loaded into 3D Slicer, an open-source image analysis software package (39). Slices in the image space were assigned a thickness of 210 µm (10 µm tissue thickness + 200 µm spacing). The render was produced using the default VTK GPU Ray Casting protocol from 3D Slicer version 5.6.2 with nearest-neighbor interpolation and without shading. The color mapping was synchronized to the volume module, which uses the quantitative image intensities of individual slices. As the 3D render was produced simply by stacking independent 2D DARs, no other reconstruction algorithm was required.
Early and late treatment study
For the therapy study, mice were injected with intracardiac injections of 22Rv1-Luc cells. One week after cell inoculations, mice were randomized and treated with either saline (vehicle) or [^225^Ac]Macropa-PEG4-YS5 at a 4.6 kBq or 9.2 kBq activity dose (n = 10). Similarly, for assessing treatment at a later time point, 3 weeks after inoculations, mice were randomized into three groups (n = 8) for saline, 4.6 kBq, or 9.2 kBq dose treatment. The antibody mass of YS5 in 4.6 and 9.2 kBq doses was 0.74 µg and 1.5 µg, respectively. Along with the saline or [^225^Ac]Macropa-PEG4-YS5 treatment, each mouse was injected with native IgG (0.5 mg) to reduce nonspecific uptake via Fc receptors (40). Bioluminescence intensity (once a week), body weight, and overall health (thrice a week) were recorded.
Statistical analysis
The results are reported as mean ± SD, and plots were prepared with GraphPad Prism (RRID: SCR_002798). The survival analysis was reported using the log-rank (Mantel–Cox) test.
Data availability
The data presented in the current study are available within the article and Supplementary Materials or from the corresponding author upon reasonable request.
Results
Intracardiac injections of prostate cancer cells develop disseminated microtumors detected with BLI and CD46 PET scans
The method of intracardiac injection has been previously used to establish disseminated tumor models (32, 41). To study the growth rate and disseminated disease pattern, Luc-expressing prostate cancer cells (22Rv1-Luc, DU145-Luc, PC3-Luc, C42B-Luc, or LNCaP-AR-Luc) were introduced into the left ventricle of mice under ultrasound guidance (36). The growth of disseminated tumors varied depending on the cell type, as evidenced by the bioluminescence signal (Supplementary Fig. S1). PC3-Luc tumors exhibited the fastest growth, with a detectable bioluminescence signal (7.2 × 10^6^ p/seconds/cm^2^/sr) at week 1 after intracardiac injections, as shown in Fig. 1A; Supplementary Fig. S1. Subsequently, DU145-Luc and 22Rv1-Luc tumors became detectable at weeks 2 and 3 after inoculations, whereas the relatively slower-growing C42B-Luc and LNCaP-AR-Luc were evident at weeks 4 and 7, respectively (Fig. 1A; Supplementary Fig. S1). The synthesis and purification of [^89^Zr]DFO-YS5, as well as the µPET/CT imaging, were conducted as previously described (23). Supplementary Fig. S2A and S2B show the instant thin-layer chromatography analysis for radiolabeled [^89^Zr]DFO-YS5, confirming the 100% purity of the product.
![Figure 1.: Identification of disseminated microtumor growth for prostate cancer cell lines using in vivo and ex vivo BLI and µPET/CT with [^89^Zr]DFO-YS5. A, Measurements of whole-body bioluminescence (radiance) for 22Rv1-Luc, PC3-Luc, DU145-Luc, C42B-Luc, and LNCaP-AR-Luc cells inoculated with intracardiac injections (n = 4). B, The BLI image, CD46 PET scan (4 days after injection of [^89^Zr]DFO-YS5), and corresponding ex vivo analysis at week 5 after inoculations for 22Rv1-Luc cells (n = 4). **C, **Ex vivo analysis showing BLI and CD46 PET maximum intensity projection (MIP) images for 22Rv1-Luc, PC3-Luc, Du145-Luc, and C42B disseminated tumor models. The BLI imaging of disseminated tumor models was correlated to the µPET/CT images and ex vivo analysis. Regions of interest were drawn over each animal to calculate the change in radiance intensity over the period. D, The BLI and µPET/CT imaging with [^89^Zr]DFO-YS5 conducted at week 1 after inoculations revealed that tumors were not detectable with low uptake (6.3% IA/g) of the probe in the liver (n = 5). E, BLI and µPET/CT imaging with [^89^Zr]DFO-YS5 performed at week 3 after inoculations displayed increased uptake of the probe in the liver (13.0 ± 1.8 %IA/g, n = 5). F, The H&E image, along with IF staining for CD46-positive cells, confirmed the presence of microtumors in the liver tissue, as observed in the digital autoradiograph for [^225^Ac]Macropa-PEG4-YS5 (n = 2). The 22Rv1-Luc inoculated mice were injected with [^225^Ac]Macropa-PEG4-YS5 3 weeks after inoculation. Livers from these mice were sliced for autoradiography, histology, and IF staining.](ccr-24-2850_f1.jpg)
The µPET/CT imaging of disseminated tumors was performed 4 days after injections of [^89^Zr]DFO-YS5. In the case of 22Rv1-Luc cell–inoculated mice, in vivo BLI and µPET/CT (Fig. 1B; Supplementary Fig. S3; Supplementary Table S1), as well as ex vivo BLI and µPET/CT (Fig. 1C), indicated the highest tumor burden in the liver 5 weeks after inoculation. In PC3-Luc and DU145-Luc tumor models (Fig. 1C; Supplementary Figs. S4 and S5), both BLI and µPET/CT scans revealed growth in the liver and surrounding areas 5 weeks after inoculation. Ex vivo BLI, µPET/CT scans, and biodistribution studies showed tumor growth in the kidneys and lungs (Fig. 1C; Supplementary Tables S2 and S3). The comparatively slow-growing C42B-Luc showed the highest involvement in the liver, followed by the lungs (Fig. 1A and C; Supplementary Fig. S6; Supplementary Table S4). Similarly, LNCaP-AR-Luc tumors reached detectable growth at week 7 after inoculation, with BLI images and µPET/CT scans suggesting liver involvement primarily (Fig. 1A; Supplementary Fig. S7). Quantifying BLI intensity from a whole-mouse image proved to be an effective method for tracking overall tumor growth (Fig. 1A). Regardless of cell type, mice with a BLI intensity of 10^8^ p/seconds/cm^2^/sr showed diarrhea, reduced locomotion, and visible bulging noted in the liver area, necessitating euthanasia. Biodistribution studies with [^89^Zr]DFO-YS5 confirmed probe uptake in the liver, with %IA/g (percent injected activity per gram) values of 22.9 ± 2.2, 14.9 ± 2.0, 12.0 ± 3.7, and 5.4 ± 2.6 for 22Rv1-Luc, PC3-Luc, DU145-Luc, and C42B-Luc, respectively (Supplementary Fig. S8). Control, nontumor-bearing mice scanned with [^89^Zr]DFO-YS5 exhibited lower probe uptake in the liver (6.8% ± 1.5 %IA/g), kidneys (4.8 ± 3.5 %IA/g), or lungs (4.3 ± 2.4 %IA/g; Supplementary Fig. S9). Disseminated tumors formed in 100% of mice injected with PC3, DU145, and 22Rv1, whereas 75% and 50% of mice showed growth of C42B and LNCaP-AR tumors, respectively. Among all the tested cell lines, 22Rv-Luc exhibited consistent and highest tumor burden in the liver with optimum growth rate, motivating subsequent studies focusing on 22Rv1-Luc model.
Disseminated tumors from intracardiac injections were detectable 3 weeks after injection
As shown in Fig. 1B and Supplementary Fig. S3, intracardiac injections of 22Rv1-Luc resulted in the development of consistent tumors, primarily in the liver, where liver tumors were detected at week 3 after inoculation, and tumor burden was highest at week 5. Therefore, we used this tumor model to observe the progression of hepatic tumor growth at both week 1 (when no BLI signal was recorded) and week 3 (when the BLI signal started to appear), utilizing [^89^Zr]DFO-YS5 and [^225^Ac]Macropa-PEG4-YS5. At weeks 1 and 3 after inoculation, mice received injections of [^89^Zr]DFO-YS5 for µPET/CT imaging. BLI imaging performed 1 week after inoculation did not show lesions, whereas the liver tumors were detectable at week 3 (Supplementary Fig. S10). The CD46 PET scan at week 1 after inoculation (Fig. 1D) revealed no specific uptake in the livers or any other tissues, with a recorded value of 6.4 ± 1.3 %IA/g in the liver compared with 6.8% ± 1.5 %IA/g in NSG control (refer to Supplementary Fig. S11; Supplementary Table S5). In contrast, the µPET/CT scan conducted 3 weeks after the intracardiac injection, when BLI signals first appeared, demonstrated an increased accumulation of [^89^Zr]DFO-YS5 in the liver, with a measured uptake of 13.0 ± 1.8 %IA/g (Fig. 1E; Supplementary Figs. S10 and S11; Supplementary Table S6). The liver uptake of [^89^Zr]DFO-YS5 continued to increase, eventually reaching 22.9 ± 2.2 %IA/g at week 5 (Supplementary Fig. S12).
Using high-resolution digital autoradiography, we then used the radioimmunotherapy agent labeled with ^225^Ac ([^225^Ac]Macropa-PEG4-YS5, radiolabeling, and purification details shown in Supplementary Fig. S13A and S13B) to investigate its distribution within the liver. In 1 week after intracardiac inoculation tumors, the distribution of [^225^Ac]Macropa-PEG4-YS5 was found to be low (2.6 ± 0.6 %IA/g) in the liver (Supplementary Fig. S14A). Autoradiography (Supplementary Fig. S14B), H&E histology (Supplementary Fig. S14C), and IF for CD46 staining (Supplementary Fig. S14D) in livers from [^225^Ac]Macropa-PEG4-YS5–treated mice confirmed the absence of probe accumulation or detectable CD46-positive cells in the liver. In contrast, the uptake of [^225^Ac]Macropa-PEG4-YS in a 3-week postinoculated tumor model was 11.4 ± 4.2 %IA/g, significantly higher than in the week 1 tumor model (Supplementary Fig. S15; Supplementary Table S7). Digital autoradiography (Fig. 1F) of the liver tissues performed on day 4 after injections revealed the accumulation of [^225^Ac]Macropa-PEG4-YS5 in the same regions (Fig. 1F), which corresponded to the differential dye uptake in H&E images (Fig. 1F) for tumor lesions. Subsequent IF staining of the liver tissue affirmed the presence of CD46-positive disseminated 22Rv1-Luc tumors, aligning with the corresponding regions seen on autoradiograph and H&E images. High-magnification views of H&E and IF images revealed distinct, spherical microtumors within the liver tissue (Fig. 1F).
Microscale dosimetry of the disseminated tumors
As the microtumors of 22Rv1-Luc were predominantly distributed in the liver, we utilized alpha-particle digital autoradiography to investigate dose deposition and activity distribution in the liver tissues. Liver tissues from mice injected with a 4.6 kBq activity were collected at 96 hours after injection; the whole liver was sectioned with a 200 µm distance between 10-µm sections for subsequent analysis (19 slices total; Supplementary Fig. S16). Figure 2A displays a representative absorbed dose-rate map illustrating the distribution of [^225^Ac]Macropa-PEG4-YS5 in the liver and microtumors. Coregistration of the dose-rate map with the H&E images (Fig. 2B) of consecutive sections confirmed the uptake of [^225^Ac]Macropa-PEG4-YS5 in 22Rv1-Luc microtumors within the liver (Fig. 2C).
![Figure 2.: Microscale dosimetry analysis for disseminated tumors of 22Rv1-Luc cells in the livers of mice treated with [^225^Ac]Macropa-PEG4-YS5. A, Dose-rate map for lesions in the liver generated from quantitative alpha-particle digital autoradiography (n = 2 animals). B, H&E image generated from the nearby tissue section for autoradiography (n = 2 animals). C, Coregistration of the dose-rate map and H&E image (n = 2 animals). D, Stacking of 2D images of H&E and dose-rate maps to generate 3D images. The tissue sections of the complete liver with microtumors were collected 200 µm apart for autoradiography and histologic staining. All the H&E images were segmented for normal liver tissue (green) and microtumors (red). The segmented images were stacked to generate the 3D image for tumor lesions. A 3D dose-rate map shows the microtumor-targeted dose in the complete volume of the liver (n = 2 animals). E, Dose-rate volume histogram summarizing all voxels in all slices of the liver. F, The microtumor (H&E) with heterogeneous dose-rate distribution resulted in lower H2AX (DNA damage) foci. G, The uniform distribution of radiation in the microtumor shows increased DNA damage.](ccr-24-2850_f2.jpg)
The 2D H&E liver-tumor masks and dose-rate maps were Z-stacked to generate a 3D view of the liver and microtumors within (Fig. 2D). The 3D renderings of histology and dose-rate maps in Fig. 2D illustrate that [^225^Ac]Macropa-PEG4-YS5 penetrated the liver tissue to distribute the alpha-particle agent even at a depth of 10 to 20 mm. Dose-rate volume histograms (Fig. 2E) summarize the calculated absorbed dose rate in the lesion (microtumor) and nonlesion areas, revealing mean values of 231 ± 153 and 11 ± 35 mGy/hour across the 3D volume, respectively—significantly higher in the microtumors. Assuming the spherical shape of microtumors, the diameter and volume of the sample microtumor in Fig. 2F were determined to be 863 µm and 0.34 mm^3^, respectively. When considering all lesions, the microtumor diameter range was between 0.1 and 1.8 mm. The total amount of radioactivity deposited in the sample microtumor cross-section, as depicted in Fig. 2F, was measured to be 608.9 mBq (0.016 nCi), corresponding to a mean activity of 0.107 mBq (2.9E-03 pCi) per cell.
Some tumors showed relatively uniform intratumoral dose deposition, whereas others demonstrated heterogeneous dose deposition, with central areas of reduced radiopharmaceutical accumulation. The heterogeneous distribution observed in the dose rate and line profiles depicted in Fig. 2F was further correlated with the presence of DNA damage foci. As illustrated in Fig. 2F, the heterogeneous dose rate resulted in a lower amount of DNA damage compared with the relatively uniform dose-rate distribution (Fig. 2G; Supplementary Fig. S17). Although the analysis of a single liver tissue slice revealed variation in deposited dose rate and corresponding DNA damage response, examination of additional slices taken 200 µm apart provided comprehensive insights into the heterogeneity within lesions (Supplementary Fig. S18). In most large lesions, the activity distribution was a thick spherical shell, uniform in edge slices but reduced in the tumor core (Supplementary Figs. S18 and S19). In contrast, small-diameter tumors received a relatively uniform radiation dose and DNA damage (Supplementary Figs. S19 and S20). We compared DNA damage events in microtumors with heterogeneous versus homogeneous dose deposition. In three microtumors with heterogeneous doses (shown in Supplementary Fig. S21A), the regions with higher doses induced 6.1 (Supplementary Fig. S21B), 7.5 (Supplementary Fig. S21C), and 8.1 (Supplementary Fig. S21D) H2AX foci per cell, respectively. Conversely, the microtumor core regions with lower radiation doses showed reduced numbers of H2AX 2.1, 3.0, and 3.6 foci per cell. Similarly, in three microtumors with homogeneous dose deposition (Supplementary Fig. S22A), the DNA damage was more uniform, with averages of 9.2 (Supplementary Fig. S22B), 6.3 (Supplementary Fig. S22C), and 8.5 (Supplementary Fig. S22D) foci at the microtumor core, respectively. Irrespective of lesion size, the crossfire from alpha particles causing DNA damage in the surrounding liver tissue was minimal (Fig. 3). A representative image of H2AX foci and DAPI staining at the tumor–liver interface (Fig. 3) shows that the first layer of hepatocytes surrounding the tumor exhibited an average of 2.1 foci per nucleus, whereas the second layer had 1.4 foci per cell. In contrast, tumor cells demonstrated significantly higher DNA damage, with an average of 14.1 foci per nucleus (Fig. 3).
![Figure 3.: DNA damage analysis (γ-H2AX foci) to study the crossfire effect on nearby liver cells following the delivery of [^225^Ac]Macropa-PEG4-YS5 to the microtumors (n = 2 animals). The livers from mice treated with [^225^Ac]Macropa-PEG4-YS5 were sectioned and stained for phospho-γ-H2AX. Liver cells immediately adjacent to tumor cells show an average of 2.1 foci per cell, whereas cells approximately 20 µm away from tumor cells exhibit an average of 1.4 foci per cell.](ccr-24-2850_f3.jpg)
The dose deposition was further tested on days 7, 10, and 14 after [^225^Ac]Macropa-PEG4-YS5 injections in 3-week-old disseminated tumors of 22Rv1-Luc (Supplementary Fig. S23). The homogeneous distribution in smaller microtumors compared with the heterogeneous distribution of the radiation dose in larger microtumors seen in the day 4 microdosimetry experiment persisted when the livers were collected on days 7, 10, and 14 after injections of [^225^Ac]Macropa-PEG4-YS5 (Supplementary Fig. S24). Over the 10 days, the mean absorbed dose rate in microtumors decayed exponentially with an effective half-life of 5.29 ± 0.05 days (Supplementary Fig. S25).
[^225^Ac]Macropa-PEG4-YS5 shows tumor cell–killing action in in vitro studies
To study the effect of [^225^Ac]Macropa-PEG4-YS5 in vitro, cell-binding and cell viability assays were performed. First, 22Rv1 and CD46 KO 22Rv1 (22Rv1-CD46KO) cells were incubated with [^225^Ac]Macropa-PEG4-YS5 for 1 hour, and the percentage of binding was determined (Fig. 4A). 22Rv1 cells showed 3.1% ± 0.1% binding, which reduced to 0.31% ± 0.1% for CD46 KO cells, whereas blocking with cold YS5 showed 0.34% ± 0.1% binding (Fig. 4A). Similarly, to study the long-term effect of [^225^Ac]Macropa-PEG4-YS5, 22Rv1 and 22Rv1-CD46KO were treated with [^225^Ac]Macropa-PEG4-YS5 for 96 hours, and cells were incubated for 2 weeks to assess colony formation. 22Rv1 cells showed a dose-dependent reduction in the number of colonies (with IC50 of 14.06 ± 0.37 Bq/mL), in which no colonies were seen in 185 or 370 Bq/mL activity–treated wells (Fig. 4B; Supplementary Fig. S26). In contrast, due to the low antibody uptake in 22Rv1-CD46KO cells, no significant reduction in colonies was observed (Fig. 4B; Supplementary Fig. S26). The DNA damage (phospho-γ-H2AX) foci studied for the various activity treatments on 22Rv1 cells showed dose-dependent DNA damage for 96 hours of treatment (Fig. 4C; Supplementary Fig. S27). Untreated samples showed 0.6 foci per cell nucleus, which gradually increased with increased radioactivity up to 16.6 foci per cell for 370 nCi/mL treatment (Fig. 4C; Supplementary Fig. S27).
![Figure 4.: Assessment of the binding and cytotoxic effect of [^225^Ac]Macropa-PEG4-YS5 on monolayer and 3D spheroids of 22Rv1 cells. A, Cell binding of [^225^Ac]Macropa-PEG4-YS5 was higher in CD46-expressing cells, as opposed to CD46-null cells and with cold YS5 blocking samples (in triplicate). B, Colony-forming assay results showing a significant cell-killing effect of [^225^Ac]Macropa-PEG4-YS5 on 22Rv1 cells compared with CD46 KO cells (treatment in triplicate). C, Increase in the number of DNA damage (phospho-γ-H2AX) foci with increasing amounts of [^225^Ac]Macropa-PEG4-YS5 on 22Rv1 cells. Three regions of >5 cells were imaged, and the average foci were calculated. D, The viability assay shows dose-dependent cell viability reduction following [^225^Ac]Macropa-PEG4-YS5 (96 hours treatment) on monolayer and 3D spheroids of 22Rv1 cells. E, The digital autoradiograph showing a heterogeneous distribution of [^225^Ac]Macropa-PEG4-YS5 in 3D spheroids. F, Z-stacking of phospho-γ-H2AX foci analysis performed on spheroids treated with [^225^Ac]Macropa-PEG4-YS5. Spheroids treated with [^225^Ac]Macropa-PEG4-YS5 for 96 hours showed the presence of phospho-γ-H2AX foci.](ccr-24-2850_f4.jpg)
In addition to testing on 2D monolayer cells, [^225^Ac]Macropa-PEG4-YS5 was also tested on 3D spheroids of 22Rv1 cells for killing and DNA damage potential. The viability assay presented in Fig. 4D showed a lower IC50 for monolayers (IC50 of 0.1 ± 0.003 kBq/mL) compared with 3D spheroids (IC50 of 1.1 ± 0.08 kBq/mL). Digital autoradiography performed on 3D spheroids treated with [^225^Ac]Macropa-PEG4-YS5 (0.4 kB/mL) showed heterogeneous distribution, in which the spheroid core received a lesser absorbed dose, similar to the observations made for microtumors growing in the livers (Fig. 4E). Subsequently, the heterogeneous distribution of radioactivity was shown to induce heterogeneous DNA damage events inside the 3D spheroids, compared with the untreated control (Fig. 4F; Supplementary Fig. S28).
Alpha-particle therapy of disseminated tumors at an early disease stage results in a CR
Two treatment strategies were implemented to assess the therapeutic response of [^225^Ac]Macropa-PEG4-YS5 in disseminated tumors of 22Rv1-Luc tumor models (Fig. 5A). The initial approach involved early treatment, with [^225^Ac]Macropa-PEG4-YS5 administered to mice at week 1 after inoculations. In both early- and late-stage treatments, mice received saline, 4.6 kBq, or 9.2 kBq of [^225^Ac]Macropa-PEG4-YS5. As seen from the BLI scans (Fig. 5B) and quantification of the BLI signals (Fig. 5C), tumor growth reached a maximum at week 5 after inoculations in saline control mice. The tumor burden was exceptionally high in the saline group, leading to mortality after week 5. The [^225^Ac]Macropa-PEG4-YS5–injected treatment groups (4.6 and 9.2 kBq) showed no growth of 22Rv1-Luc tumors. The median survival of the saline group was 7 weeks, whereas 9 out of 10 mice in both the 4.6 and 9.2 kBq groups survived until the endpoint of the study (week 17; Fig. 5C). One mouse from each of the 4.6 and 9.2 kBq treatment groups showed mortality at week 15 without any visible symptoms of toxicity. The body weights of the mice in the radioactivity-injected groups decreased by 12% in the first 2 weeks of activity injections, but all the mice regained their body weights, experiencing continuous gain until week 17 (Supplementary Fig. S29). Subsequently, we conducted µPET/CT scans using [^89^Zr]DFO-YS5 to verify the presence of residual disseminated disease. The scans shown in Fig. 5D and E, along with the biodistribution study (Supplementary Fig. S30; Supplementary Table S8), confirmed a low liver uptake of the probe in mice treated with both the 4.6 kBq (5.33 ± 0.79 %IA/g) and 9.2 kBq activity (6.69 ± 0.99 %IA/g). Given that 22Rv1-Luc tumors grow in the liver, we collected liver samples from the mice in the treatment groups and conducted histologic examinations and IF staining for CD46. The results obtained from H&E images and IF images further confirmed the absence of growth of 22Rv1-Luc cells (Fig. 5D and E).
![Figure 5.: Treatment of the 22Rv1-Luc disseminated tumor model with [^225^Ac]Macropa-PEG4-YS5 at an early stage completely eliminates the disease. A, Schematic representation of the experimental plan for early treatment studies. Treatment studies were performed 1 week following intracardiac injection of 22Rv1-Luc cells. B, BLI scans for the mice injected with saline, 4.6 kBq, and 9.2 kBq activity of the [^225^Ac]Macropa-PEG4-YS5 at week 1 after intracardiac injections of the cells. CR was observed in 100% of the mice in the [^225^Ac]Macropa-PEG4-YS5 treatment group (n = 10 per arm). C, Quantification of the BLI signal from images revealing continuous tumor growth in saline-treated mice, whereas [^225^Ac]Macropa-PEG4-YS5–treated mice showed an absence of BLI. The Kaplan–Meier curve depicts early mortality in the saline control groups with a significant improvement in [^225^Ac]Macropa-PEG4-YS5–treated cohorts (n = 10 per arm). ****, *P *< 0.0001. D, The µPET/CT scans for a 4.6 kBq dose in early treatment, liver histology, and CD46 IF for tumor cell detection. The scans performed 4 days after injection of [^89^Zr]DFO-YS5 showed no specific uptake of the probe, whereas H&E and IF showed an absence of tumor cells (n = 4 per arm). E, CD46 PET/CT, histologic staining, and IF staining of the livers from mice in 9.2 kBq activity groups confirm CR and absence of tumor lesions (n = 4 per arm).](ccr-24-2850_f5.jpg)
Treatment of disseminated disease at a late stage requires higher administered activity to achieve a CR
Subsequently, we initiated treatment 3 weeks after intracardiac inoculations of 22Rv1-Luc (Fig. 6A). The bioluminescence signal at week 3 was significantly higher than the early treatment’s starting point. Figure 6B and C provide a comparison of the BLI scans from week 3 to week 17. As anticipated, the saline group exhibited rapid tumor growth, resulting in 7 of 8 mice mortality in the sixth week (Fig. 6B). In the case of the 4.6 kBq activity treatment group, we observed an initial decrease in BLI signal, indicating tumor growth inhibition until week 8 (Fig. 6B and C). Following week 8, mice in the 4.6 kBq activity treatment group showed increased BLI intensity corresponding to tumor growth (Fig. 6B and C). Two of eight mice from the 4.6 kBq activity treatment group showed a CR. In contrast, the bioluminescence intensity decreased significantly to the background level in the 9.2 kBq group. This suggests a CR as soon as week 6 (3 weeks after dose injections) of cell injections (Fig. 6C). The median survival of the saline and 4.6 kBq treatment was found to be 6 and 16.5 weeks (P < 0.0001), respectively (Fig. 6C). Six of eight mice survived in the 9.2 kBq activity treatment group at the end of the experiment. At week 17 after intracardiac inoculations, mice were injected with [^89^Zr]DFO-YS5 for µPET/CT scans. The BLI signal and CD46 PET scans were positive for one mouse from the 4.6 kBq treatment group (Fig. 6D). Further histology and IF staining confirmed the presence of CD46-positive 22Rv1-Luc cells in the lungs, but the liver from the same mouse was clear of disease [Fig. 6D (liver inset)]. In contrast, mice from the 9.2 kBq treatment did not show any sites of abnormal uptake, suggesting the complete eradication of the tumor (Fig. 6E; Supplementary Fig. S31; Supplementary Table S9). The CD46 IF staining and histology carried out for the mice from the 9.2 kBq activity group corroborate the absence of 22Rv1 cells or tumors (Supplementary Fig. S32).
![Figure 6.: Treatment of the 22Rv1-Luc tumor models at the late stage of the disease requires a higher administered activity to completely eliminate disseminated disease. A, Schematic representation of the experimental plan for late treatment (after 3 weeks of inoculations) studies. B, Bioluminescence scans for the late treatment study in which mice were injected with saline and [^225^Ac]Macropa-PEG4-YS5 (4.6 and 9.2 kBq, n = 8 per group). C, Quantification of the BLI intensity to track the disseminated tumor growth. Total flux (radiance) in the 9.2 kBq treatment group was reduced to background level showing CR, whereas the 4.6 kBq dose showed regrowth of tumors. A Kaplan–Meier plot of mice treated with saline, 4.6 kBq, and 9.2 kBq activity of [^225^Ac]Macropa-PEG4-YS5 shows significant improvement in survival (n = 8 per group). ****, P < 0.0001. **D, **In vivo BLI, µPET/CT, and ex vivo histology and CD46 IF staining for lungs and liver for a mouse in the 4.6 kBq cohort. The regrowth of disseminated tumors seen in the lower dose of 4.6 kBq was detectable by BLI and PET/CT imaging with [^89^Zr]DFO-YS5. CD46 expressed on relapsed tissue was confirmed with CD46 IF (n = 2). E, µPET/CT images showing no uptake of the [^89^Zr]DFO-YS5 in a mouse with CR from the 9.2 kBq treatment groups (n = 3).](ccr-24-2850_f6.jpg)
Toxicity analysis of [^225^Ac]Macropa-PEG4-YS5 reveals no tissue damage at the 4.6 and 9.2 kBq dose levels
To assess the toxicity of [^225^Ac]Macropa-PEG4-YS5 in treated mice, we collected blood, serum, and tissue samples at week 17 of intracardiac inoculations. A CBC was conducted to determine red blood cells and white blood cells. Serum samples were processed to analyze major liver and kidney damage markers, including alanine transaminase, aspartate transaminase, alkaline phosphatase, blood urea nitrogen, and creatinine. The CBC analysis (Supplementary Fig. S33A) showed an increase in absolute monocyte counts in the 9.4 kBq dose (0.21 ± 0.10 × 10^3^/μL) treatment group compared with healthy control (0.09 ± 0.04 × 10^3^/μL; P < 0.01, heteroscedastic one-sided two-sample t test). The liver and kidney function tests (Supplementary Fig. S33B) revealed no changes in the parameters studied when compared with the healthy control group (labeled as saline), indicating the absence of any liver or kidney toxicity in the mice that survived from both early and late treatment groups. Alpha-particle therapy agents based on ^225^Ac are known to cause the accumulation of daughter isotopes in the kidneys, potentially leading to nephrotoxicity. As a comprehensive assessment, we processed liver, kidneys, heart, lungs, and femur samples for histologic analysis to identify microscopic changes for toxicity evaluation. No organ toxicity was associated with surviving mice injected with [^225^Ac]Macropa-PEG4-YS5 at 4.6 and 9.2 kBq activity doses (Supplementary Fig. S34). As noted in the CBC analysis, the spleens from the 9.4 kBq dose showed an increase in the monocyte levels (Supplementary Fig. S34).
Discussion
Alpha particles possess a short range and high linear energy transfer (42), resulting in higher relative biological effectiveness and greater therapeutic efficacy (43). A few decay events emitting alpha particles can cause significant, irreparable DNA damage when delivered to cancer cells using a target vector (44). Therefore, alpha-particle therapy has been recommended for use in single-cell diseases, including hematologic malignancies, circulating tumor cells, and micrometastatic disease (24, 45, 46). In this study, we present the development, dosimetry analysis, and alpha-particle treatment of disseminated models of prostate cancer. Intracardiac inoculations of prostate cancer cells were utilized to develop disseminated tumor models, in which the distribution of cells gives rise to microtumors in distant organs. PC3-Luc cells, derived from bone metastasis patient cells, show the fastest growth due to their aggressive nature and a faster doubling time (approximately 25 hours). Tracking tumor growth in all models (22Rv1-Luc, PC3-Luc, C42B-Luc, DU145-Luc, and LNCaP-AR-Luc) was conducted through BLI and µPET/CT, revealing the highest tumor burden in the liver, followed by the lungs and kidneys. The disseminated tumor growth pattern of DU145-Luc (established from central nervous metastatic patients) was similar to PC3-Luc, displaying growth in the liver, kidneys, and lungs (Fig. 1). Considering the disseminated growth pattern, PC3-Luc and DU145-Luc tumor models were the most suitable choices for alpha-particle therapy. However, the applicability of PC3-Luc and DU145-Luc to our study was limited by the presence of tumors in the kidneys. Our study aimed to investigate the off-target nephrotoxicity of [^225^Ac]Macropa-PEG4-YS5, as previous studies have demonstrated renal injury due to the release of ^213^Bi from actinium conjugates (25). The C42B-Luc (derived from bone metastasis) and LNCaP-AR (from lymph metastasis) models were also considered for targeted alpha-particle therapy; however, the lower take rate and slow growth of these tumors discouraged their selection for our therapy studies. Encouragingly, [^89^Zr]DFO-YS5 PET detected the disseminated disease in all examined cell lines, including PSMA-negative and PSMA-positive cells.
The 22Rv1 cells, isolated initially from relapsed CWR22 xenograft, showed an optimal growth rate and consistently formed liver tumors in all mice with a 100% take rate (47). Our results for 22Rv1-Luc cells growing in the liver are partly consistent with the studies reported by Stuparu and colleagues (32) and Drake and colleagues (41), in which kidney, adrenal gland, and lung involvement were noted along with the liver. We utilized intracardiac injections to develop disseminated tumors of 22Rv1-Luc for our studies. The [^89^Zr]DFO-YS5 probe uptake from CD46 µPET/CT scans and biodistribution at weeks 1, 3, and 5 after inoculations, along with posttreatment imaging, validates the probe’s efficacy in studying tumor progression and treatment response. Disseminated tumor growth of 22Rv1-Luc cells was not detectable at week 1 after inoculation, but they reached a size detectable by BLI and µPET/CT at week 3 after inoculations.
We tested the alpha-particle therapy agent [^225^Ac]Macropa-PEG4-YS5 on disseminated tumors of 22Rv1-Luc cells. Even though ^225^Ac produces detectable γ-ray emissions, the low activity levels of injections present difficulties in conducting in vivo imaging and image-based dosimetric evaluations (48). We instead performed ex vivo microdosimetry analysis of the liver and microtumors using digital alpha-particle autoradiography (37) and correlated it with adjacent liver sections stained for histology and DNA damage to provide insights into radiobiological effects. The microtumors in the liver vary in size up to a diameter of 1.8 mm, whereas varying degrees of absorbed dose were deposited in the microtumors (100–550 mGy/hour). The low total absorbed dose in liver tissue and the higher dose in microtumors resulted in significantly higher DNA damage in tumor cells. Liver microtumors that were uniformly dosed resulted in higher DNA damage compared with heterogeneously dosed microtumors, supporting the hypothesis of the utilization of alpha-particle therapy for micrometastatic disease (46). The microtumor core with a lower absorbed dose showed a lesser number of H2Ax foci. Interestingly, dose distribution was not fully spatially colocalized in the tumor with CD46 expression. A variety of mechanisms may account for this, including heterogeneous vasculature, heterogeneous enhanced permeability and retention effect, or the binding site barrier effect (49–51). One additional limitation of this study was the small number of mice used for the dosimetry analysis, owing to the complexity of the sectioning and measurement procedures. In order to address these important scientific questions, we plan a future study incorporating different types of radiopharmaceuticals in this disseminated microtumor model, incorporating a larger number of mice.
Our findings underscore the effectiveness of treating early-stage disease, characterized by the absence of detectable BLI signals, compared with later stages, in which the tumor burden was significantly higher. Early treatment of 22Rv1-Luc with [^225^Ac]Macropa-PEG4-YS5 resulted in a CR in both treatment arms (4.6 and 9.2 kBq). Similarly, late treatment with a higher dose (9.2 kBq) resulted in a CR in 100% of animals, whereas the lower dose showed regrowth in 75% of animals. These findings in treatment studies are in accordance with findings reported by Stuparu and colleagues (32) for the treatment of disseminated tumor model of C4-2 cells. Stuparu and colleagues showed that early treatment with ^225^Ac-PSMA-617 prevented liver dissemination, whereas late treatment enhanced survival at advanced stages of the disease. This is particularly relevant as recent studies are investigating the potential of RLT as an earlier line of therapy (52, 53). Additionally, patients with prostate cancer with liver metastases have shown decreased PSMA expression compared with other metastatic sites. Therefore, [^225^Ac]Macropa-PEG4-YS5 treatment could be a significant therapeutic strategy for targeting liver metastases (54). CD46 is weakly expressed in normal human liver tissue, allowing the YS5 antibody, which targets a cancer-specific epitope, to provide more precise treatment of liver metastasis without significant off-target effects.
Late treatment significantly diminishes the likelihood of successful treatment, necessitating more aggressive dosing strategies. Smaller lesions in the liver received a more uniformly distributed absorbed dose and increased DNA damage, and early-stage tumors not detectable by µPET/CT scan or BLI were effectively treated even at the lower 4.6 kBq injected activity. In contrast, during the late stage, treatment with the low activity dose group initially showed a decrease in tumor growth until week 8; however, subsequent tumor regrowth indicates that higher doses might be necessary to sustain tumor control. The heterogeneous deposition of the radiopharmaceutical may be due to inadequate penetration of the antibody.
The NSG mouse strain used in our experiments, which carries the Prkdc^scid^ mutation, is sensitive to radiation (55). This sensitivity results in enhanced toxicities at higher doses. Our earlier study demonstrated that an 18.5 kBq dose led to the highest antitumor response for subcutaneous 22Rv1 tumors (25); however, the accumulation of free ^213^Bi in the kidneys resulted in elevated creatinine levels, and histologic analysis revealed damage to the glomeruli (25). Similarly, the death of three mice in the late-treatment strategy for a 9.2 kBq dose indicates a form of toxicity that is not visibly apparent and does not result in body weight loss. A limitation of our study is that we did not perform necropsy on the mice that died at weeks 10 and 12 after inoculation, which could have provided important information about toxicity. The mice surviving at the end of our study (week 17 after inoculations) show no toxicity to the kidneys or any other organs. Our quantitative digital autoradiography and corresponding DNA damage analysis (Figs. 2 and 3) are consistent with the reduced crossfire effect of alpha particles on healthy tissues, owing to their shorter ranges. We observed that a layer of liver cells within a 10 µm distance from the microtumor exhibited slightly increased DNA damage, whereas cells at a 20 µm distance showed reduced damage. Given that alpha particles have a maximum travel distance of approximately 100 µm, the crossfire effect on liver cells was minimal.
The promising outcomes of alpha-particle therapy on disseminated tumor models support the future clinical application of [^225^Ac]Macropa-PEG4-YS5 for metastatic prostate cancer. The promise of this ^225^Ac-based radioimmunotherapy is underscored by recently published favorable phase I results of the PSMA-targeting ^225^Ac-J591 (5). [^225^Ac]Macropa-PEG4-YS5 effectively targets the overexpressed CD46 on prostate cancer tumors. Ongoing clinical trials involving our CD46-targeting ADC and PET imaging probes seek to further validate this therapeutic approach (NCT03575819, NCT05011188, and NCT05245006; refs. 17, 23). Additionally, our recent advancements in [^225^Ac]DOTA-YS5 (24, 25) and [^225^Ac]Macropa-PEG4-YS5 (27, 28) represent significant progress toward translating anti-CD46 alpha-particle therapy agents into clinical use.
In conclusion, we have developed various disseminated models of prostate cancer to aid in preclinical testing of radiopharmaceuticals. Due to the specificity of the [^225^Ac]Macropa-PEG4-YS5 for CD46 on 22Rv1 cells, we delivered high radiation doses to microtumors growing in the livers. The targeted delivery of the alpha-particle agent [^225^Ac]Macropa-PEG4-YS5 showed therapeutic efficacy against disseminated tumor models although heterogeneous dose deposition reduced efficacy against larger lesions. The data presented in this study provide support for the clinical application of [^225^Ac]Macropa-PEG4-YS5 in treating metastatic tumors.