177Lu Anti-Angiogenic Radioimmunotherapy Targeting ATP Synthase in Gastric Cancer Model

This study investigated a novel radioimmunotherapy strategy for targeting tumor angiogenesis. We developed a radiopharmaceutical complex by labeling an anti-adenosine triphosphate synthase (ATPS) monoclonal antibody (mAb) with the radioisotope 177Lu using DOTA as a chelating agent. 177Lu-DOTA-ATPS mAb demonstrated high labeling efficiency (99.0%) and stability in serum. MKN-45 cancer cells exhibited the highest cellular uptake, which could be specifically blocked by unlabeled ATPS mAb. In mice, 177Lu-DOTA-ATPS mAb accumulated significantly in tumors, with a tumor uptake of 16.0 ± 1.5%ID/g on day 7. 177Lu-DOTA-ATPS mAb treatment significantly reduced the viability of MKN-45 cells in a dose-dependent manner. In a xenograft tumor model, this radioimmunotherapy strategy led to substantial tumor growth inhibition (82.8%). Furthermore, combining 177Lu-DOTA-ATPS mAb with sunitinib, an anti-angiogenic drug, enhanced the therapeutic efficacy of sunitinib in the mouse model. Our study successfully developed 177Lu-DOTA-ATPS mAb, a radioimmunotherapy agent targeting tumor blood vessels. This approach demonstrates significant promise for inhibiting tumor growth, both as a single therapy and in combination with other anti-cancer drugs.


Introduction
Angiogenesis, the formation of new blood vessels, is a crucial process for normal tissue growth and tumor expansion.Disrupting this process has become a major focus for researchers aiming to develop effective cancer therapies.Tumors hijack angiogenesis to fuel their growth and metastasis [1].This uncontrolled vessel formation is countered by endogenous inhibitors like angiostatin.Angiostatin maintains a balanced angiogenic environment by suppressing the effects of proangiogenic factors like vascular endothelial growth factor and fibroblast growth factor [2].While typically residing within the mitochondrial inner membrane, adenosine triphosphate synthase (ATPS), an enzyme responsible for ATP generation, has been found on the surface of endothelial and tumor cells, called "ectopic" ATPS [3].Specific subunits (α/β) of this ectopic ATPS serve as binding sites for angiostatin [4].Antibodies developed against specific ATPS subunits can directly inhibit the enzyme's activity on the endothelial cell surface, which, in turn, disrupts the formation of new blood vessels and directly hinders the proliferation and migration of cancer cells [5][6][7].We also have shown that radiolabeled anti-ATPS antibodies are promising tools for radioimmunotherapy and immuno-positron emission tomography (PET) imaging.Radioiodine ( 131 I)-labeled anti-ATPS antibody effectively suppressed the tumor growth by 2.5-fold in a gastric cancer model [8].Anti-ATPS antibodies labeled with Zirconium-89 ( 89 Zr) demonstrated significantly higher targeting specificity for MDA-MB-231 tumors with abundant ectopic ATPS expression compared to PC-3 tumors with low expression [9].Given its role in tumor angiogenesis, ectopic ATPS emerges as a novel target for developing effective anti-angiogenic therapies.
Since the landmark report in 1981 on a successful radiolabeled antibody targeting carcinoembryonic antigen [10], radioimmunotherapy has remained a beacon of hope in the fight against cancer, offering a powerful tool for targeted therapy.Zevalin™ and Bexxar™, being used for the treatment of non-Hodgkin's lymphoma, are prime examples of radioimmunotherapy successfully employed in modern medicine.Lutetium-177 ( 177 Lu) has gained widespread popularity as a preferred radioisotope in recent years.This radioisotope demonstrates its versatility in cancer treatment.Studies have shown its effectiveness in treating two distinct cancers: unresectable metastatic neuroendocrine tumors [11] and metastatic castration-resistant prostate cancer [12].In both cases, it is used as a targeted therapy approach (peptide receptor radionuclide therapy and radioligand therapy). 177Lu has favorable characteristics for radioimmunotherapy.It emits both high-energy β-ray (Emax = 761 KeV) and γ-rays (113 in 6.4% and 208 KeV in 13%) and decays with a half-life of 6.7 days [13], which is favorable for radioimmunotherapy.
This study aims to develop a new radioimmunotherapy approach that specifically targets tumor angiogenesis.We synthesized 177 Lu-labeled anti-ATPS antibody to achieve this and evaluated its antitumor efficacy in a gastric cancer animal model.
Since the landmark report in 1981 on a successful radiolabeled antibody targeting carcinoembryonic antigen [10], radioimmunotherapy has remained a beacon of hope in the fight against cancer, offering a powerful tool for targeted therapy.Zevalin™ and Bexxar™, being used for the treatment of non-Hodgkin's lymphoma, are prime examples of radioimmunotherapy successfully employed in modern medicine.Lutetium-177 ( 177 Lu) has gained widespread popularity as a preferred radioisotope in recent years.This radioisotope demonstrates its versatility in cancer treatment.Studies have shown its effectiveness in treating two distinct cancers: unresectable metastatic neuroendocrine tumors [11] and metastatic castration-resistant prostate cancer [12].In both cases, it is used as a targeted therapy approach (peptide receptor radionuclide therapy and radioligand therapy). 177Lu has favorable characteristics for radioimmunotherapy.It emits both high-energy βray (Emax = 761 KeV) and γ-rays (113 in 6.4% and 208 KeV in 13%) and decays with a halflife of 6.7 days [13], which is favorable for radioimmunotherapy.
This study aims to develop a new radioimmunotherapy approach that specifically targets tumor angiogenesis.We synthesized 177 Lu-labeled anti-ATPS antibody to achieve this and evaluated its antitumor efficacy in a gastric cancer animal model.
All cells except FTC-133 (FTC-133 cells were cultured in DMEM/F-12, 1:1 mixture, 10% fetal bovine serum, WelGENE Inc., Daegu, Republic of Korea) were cultured with RPMI-1640 medium (WelGENE) supplemented with 10% FBS and 1% penicillin/streptomycin (WelGENE) at 37 • C and 5% fully humidified CO 2 .Animal experiments were performed according to protocols approved by the Care of Experimental Animals Committee (IACUC No. 2021-0067).Six-week-old female Balb/c nude mice (Orient Bio, Seongnam, Republic of Korea) were maintained under specific pathogen-free conditions [15].To create a tumor xenograft model, 5 × 10 6 tumor cells were mixed with phenol red-free Matrigel (Corning ® , Bedford, MA, USA) and subcutaneous inoculation was injected into the right shoulder of each mouse.Experiments were performed about 10 to 14 days after injection of cells, when tumors reached a diameter of approximately 5 to 10 mm.

Cellular Uptake of 177 Lu-DOTA-ATPS mAb in Various Cancer Cells
Cellular uptake of 177 Lu-DOTA-ATPS mAb was measured in the six human cancer cell lines as described previously [8].In brief, 5 × 10 5 cells were seeded per well in 12-well plates and cultured for 24 h.Upon attachment, 37 kBq 177 Lu-DOTA-ATPS mAb was added to freshly replaced culture media, followed by incubation of the cells for 1, 4, or 24 h at 37 • C and 5% CO 2 .After incubation, the cells were washed twice with cold PBS and harvested with 0.1 N NaOH.Radioactivity of the cells was counted using a Gamma-HEs gamma counter (Shinjin Medics Inc., Goyang, Republic of Korea) and normalized to the cell protein content obtained using the Bradford method [16].Data are presented as the percentage of 1 h uptake.

Specific Binding of 177 Lu-DOTA-ATPS mAb in MKN-45 Cells
MKN-45 cells were cultured in 12-well plates and incubated with 111 kBq free 177 Lu, 177 Lu-DOTA-ATPS mAb, or 177 Lu-DOTA-IgG for 24 h at 37 • C and 5% CO 2 .After incubation, the cells were washed twice with cold PBS, harvested with 0.1 N NaOH, and the radioactivity was counted using a gamma counter.The cell protein content was determined using the Pierce 660 TM Protein Assay Kit for normalization (Thermo Fisher Scientific, Rockford, IL, USA).Cellular uptake was expressed as a percentage of free 177 Lu uptake.

Competitive Binding of 177 Lu-DOTA-ATPS mAb in MKN-45 Cells
Competitive inhibition of 177 Lu-DOTA-ATPS mAb binding was examined using unlabeled ATPS mAb in MKN-45 cells.Cells were cultured in 12-well plates and treated with 10% FBS and 1% penicillin/streptomycin at 37 • C and 5% CO 2 (as described previously).The cells were pretreated with 6.4 µM unlabeled ATPS mAb for 1 h, while control cells were incubated with the vehicle.Then, 111 kBq 177 Lu-DOTA-ATPS mAb was added to the cells and incubated for 4 or 24 h under the same conditions.After incubation, cellular uptake was calculated and expressed as a percentage relative to that of untreated control.Cellular uptake, specific binding, and inhibition experiments were all performed in triplicate.

Biodistribution Study of Wild-Type Mice and MKN-45 Tumor Xenograft Models
Wild-type mice and MKN-45 tumor-bearing mice (n = 4-5 per time point) were intravenously injected with 3.7 MBq 177 Lu-DOTA-ATPS mAb, 177 Lu-IgG, or free 177 Lu.The mice were then anesthetized, sacrificed, and dissected for organ radioactivity analysis at 1, 2, 4, and 7 days after injection.Major organs (heart, lung, liver, spleen, stomach, kidneys, intestine, muscle, and bone), blood, and tumors were dissected, weighed, and counted for radioactivity using a gamma counter.Uptake in the organs and tumors was expressed as the percentage of the injected dose per gram of tissue (%ID/g).

Blocking Study of 177 Lu-DOTA-ATPS mAb in MKN-45 Tumor-Bearing Mice
To investigate blocking efficacy, 50 µg of unlabeled ATPS mAb was co-injected with 3.7 MBq 177 Lu-DOTA-ATPS mAb (1 µg as mAb) through the tail vein (n = 2).Tumors and organs were then removed at 1 and 7 days after injection for subsequent radioactivity analysis.The organs were weighed and counted for radioactivity using a gamma counter.Results were expressed as %ID/g.
The effect of combination therapy of 177 Lu-DOTA-ATPS mAb and sunitinib was evaluated in mice bearing MKN-45 tumors.Mice were divided into four groups: vehicle (0.9% NaCl), 18.5 MBq 177 Lu-DOTA-ATPS mAb alone, 40 mg/kg sunitinib alone, or a combination of both (n = 6 for each group).Mice received 18.5 MBq 177 Lu-DOTA-ATPS mAb and 40 mg/kg sunitinib once a week for 4 weeks [19].Tumor size was measured twice a week as described above.Body weights were measured once a week.
For immunohistochemistry, tumors were dissected immediately after the PET imaging at the 4th week.Slides were stained using an anti-CD31 antibody (ab28364, abcam) according to the manufacturer's standard procedure.

Statistical Analysis
All data are presented as means ± standard errors.The statistical comparison of cellular uptake and tumor size was evaluated by Student's t-test and Kruskal-Wallis test using statistical software (R, version 3.1.2),and the difference was considered significant at p < 0.05.

Labeling Efficiency and In Vitro Stability of 177 Lu-DOTA-ATPS mAb
The 177 Lu-DOTA-ATPS mAb was successfully synthesized according to the schematic representation.The radiochemical yield of 177 Lu-DOTA-ATPS mAb was 99.0% (Figure 2A).The in vitro stabilities of 177 Lu-DOTA-ATPS mAb in serum were at least 95% on the 2nd day and 85% on the 7th day at all temperature conditions (Figure 2B).Similarly, the in-vitro stabilities of 177 Lu-DOTA-ATPS mAb in PBS were at least 94% on the 2nd day, regardless of temperature.However, on the 7th day, the stability dropped significantly to 65%, 68%, and 91% at 4 • C, room temperature, and 37 • C, respectively (all p < 0.005).
representation.The radiochemical yield of 177 Lu-DOTA-ATPS mAb was 99.0% (Figure 2A).The in vitro stabilities of 177 Lu-DOTA-ATPS mAb in serum were at least 95% on the 2nd day and 85% on the 7th day at all temperature conditions (Figure 2B).Similarly, the invitro stabilities of 177 Lu-DOTA-ATPS mAb in PBS were at least 94% on the 2nd day, regardless of temperature.However, on the 7th day, the stability dropped significantly to 65%, 68%, and 91% at 4 °C, room temperature, and 37 °C, respectively (all p < 0.005).
FOR PEER REVIEW 7 of 16
For 177 Lu-DOTA-IgG, bone marrow uptake remained lower than that of 177 Lu-DOTA-ATPS mAb and 177 LuCl 3 throughout the study (all < 10%ID/g).Uptake of 177 Lu-DOTA-IgG was similar among the liver, spleen, and kidneys.Renal uptake reached a peak on day 1, while hepatic and splenic uptake increased over time.
In an inhibition study, a high dose of unlabeled ATPS mAb significantly reduced the tumoral uptake of 177 Lu-DOTA-ATPS mAb from 6.08 ± 1.0%ID/g on day 1 to 3.8 ± 1.1%ID/g (p < 0.05).

Radioimmunotherapy, Immunohistochemical Staining, and 18 F-FDG-PET Imaging in MKN-45 Tumor-Bearing Mice
All animals survived until the end of the experiment regardless of treatment (single agent or combination).
Immunohistochemistry using an anti-CD31 antibody revealed moderate to strong staining in the small vessels of the tumors treated with unlabeled ATPS mAb, IgG, or vehicle (Figure 7B).Conversely, tumors treated with 177 Lu-DOTA-ATPS mAb exhibited minimal staining.
Representative 18 F-FDG PET images of mice after four weeks of treatment are shown in Figure 7C.Tumor volume increased in mice treated with unlabeled ATPS mAb, IgG, or vehicle at the 4th week.These tumors also displayed central metabolic defects, indicative of necrotic change.In contrast, tumor volume remained stable in mice treated with 177 Lu-DOTA-ATPS mAb.
Representative 18 F-FDG PET images of mice after four weeks of treatment are shown in Figure 8C.Tumor volume increased in all groups at the 4th week.Tumors treated with single agents or vehicle were larger than those treated with the combination and displayed central metabolic defects.Immunohistochemistry using an anti-CD31 antibody revealed strong staining in the small vessels of tumors from the vehicle group only (Figure 8B).In contrast, tumors treated with 177 Lu-DOTA-ATPS mAb, sunitinib or the combination exhibited minimal staining.These findings indicate an anti-angiogenic effect of the therapeutic approaches.
Representative 18 F-FDG PET images of mice after four weeks of treatment are shown in Figure 8C.Tumor volume increased in all groups at the 4th week.Tumors treated with single agents or vehicle were larger than those treated with the combination and displayed central metabolic defects.

Discussion
In the present study, we aimed to develop a radioimmunotherapy agent targeting tumor angiogenesis.We achieved this by coupling anti-ATPS mAb to 177 Lu using DOTA as a chelator.Based on cellular uptake results, MKN-45 gastric cancer cells were selected for further development and the evaluation of the therapeutic efficacy of 177 Lu-DOTA-ATPS mAb.The uptake of 177 Lu-DOTA-ATPS mAb was specific and inhibited by unlabeled ATPS mAb in both in vitro and in vivo experiments. 177Lu-DOTA-ATPS mAb demonstrated a superior therapeutic effect compared to unlabeled ATPS mAb against MKN-45 cells, both in vitro and in vivo.Furthermore, the combination of 177 Lu-DOTA-ATPS mAb with sunitinib significantly enhanced the therapeutic effect in mice bearing MKN-45 tumors.This enhanced efficacy was also evident in 18 F-FDG PET imaging and immunohistochemistry analysis.These results suggest that radioimmunotherapy using 177 Lu-DOTA-ATPS mAb has potential for application in cancer therapy targeting tumor angiogenesis. 177Lu is a long-lived (half-life of 6.7 days), β-ray emitting (Emax = 0.49 MeV, range = 670 µm in soft tissue) radioisotope suitable for therapy.The success of 177 Lu-based radioligand therapy and peptide receptor radionuclide therapy has contributed to the grow-

Discussion
In the present study, we aimed to develop a radioimmunotherapy agent targeting tumor angiogenesis.We achieved this by coupling anti-ATPS mAb to 177 Lu using DOTA as a chelator.Based on cellular uptake results, MKN-45 gastric cancer cells were selected for further development and the evaluation of the therapeutic efficacy of 177 Lu-DOTA-ATPS mAb.The uptake of 177 Lu-DOTA-ATPS mAb was specific and inhibited by unlabeled ATPS mAb in both in vitro and in vivo experiments. 177Lu-DOTA-ATPS mAb demonstrated a superior therapeutic effect compared to unlabeled ATPS mAb against MKN-45 cells, both in vitro and in vivo.Furthermore, the combination of 177 Lu-DOTA-ATPS mAb with sunitinib significantly enhanced the therapeutic effect in mice bearing MKN-45 tumors.This enhanced efficacy was also evident in 18 F-FDG PET imaging and immunohistochemistry analysis.These results suggest that radioimmunotherapy using 177 Lu-DOTA-ATPS mAb has potential for application in cancer therapy targeting tumor angiogenesis. 177Lu is a long-lived (half-life of 6.7 days), β-ray emitting (Emax = 0.49 MeV, range = 670 µm in soft tissue) radioisotope suitable for therapy.The success of 177 Lu-based radioligand therapy and peptide receptor radionuclide therapy has contributed to the growing popularity of 177 Lu as a radioisotope for radioimmunotherapy [20]. 177Lu-PSMA has shown promising results as a treatment option for metastatic castration-resistant prostate cancer following chemotherapy and hormonal therapy [21]. 177Lu-DOTA-TATE effectively reduces tumor growth and stabilizes disease in patients with gastroenteropancreatic welldifferentiated neuroendocrine tumors, leading to its establishment as a second-or third-line treatment option [22]. 177Lu-labeled trastuzumab, an antibody that targets the HER2 receptor protein, exemplifies radioimmunotherapy using 177 Lu.This approach demonstrates specific uptake in HER2-positive primary breast cancers and their metastatic sites [23].Radioimmunotherapy with 177 Lu has also been explored in anti-angiogenesis cancer treatment. 177Lu-labeled TRC105, an antibody targeting CD105, demonstrated tumor uptake in mice with breast cancer.The uptake was 14.3 ± 2.3%ID/g on day 1 and 11.6 ± 6.1%ID/g on day 7, similar to our findings.This approach also inhibited tumor growth and improved survival [24].These results provide strong support for the use of 177 Lu-labeled radiopharmaceuticals in radioimmunotherapy.
Among the 177 Lu-labeled radiopharmaceuticals previously mentioned, 177 Lu-DOTA-TATE (Lutathera ® ) was approved by the FDA in 2018 for the treatment of somatostatin receptor-positive gastroenteropancreatic neuroendocrine tumors [25].Additionally, 177 Lu -PSMA-617 (Pluvicto ® ) was approved in 2022 for the treatment of adult patients with prostate-specific membrane antigen-positive metastatic castration-resistant prostate cancer who have previously been treated with androgen receptor pathway inhibition and taxane-based chemotherapy [26].Due to the success of 177 Lu-based therapy, it has recently garnered significant attention.In the near future, we can expect the development of more radiopharmaceutical therapies, not limited to those labeled with 177 Lu.This progress will provide clinicians with a broader range of treatment options for their cancer patients. 177Lu, a radiometal isotope, requires a chelating agent to form a stable complex with antibodies.DOTA, diethylenetriamine pentaacetate (DTPA), and ethylene-diaminetetraacetic acid (EDTA) are the most common chelators for radiometal isotopes [27,28].In our preliminary study, 177 Lu-DOTA-ATPS mAb exhibited excellent labeling efficiency (around 99.0%) and stability in repeated experiments.Conversely, the labeling efficiency of 177 Lu-DTPA-ATPS mAb was significantly lower.This aligns with previous reports [29,30].We opted for DOTA-based mAb on these findings.
DOTA, a macrocyclic chelator offers greater in vivo stability compared to acyclic chelators like DTPA and EDTA ("macrocyclic effect") [27].This translates to its recommendation for labeling 177 Lu.As shown in this study, free 177 Lu accumulates significantly in bone marrow (72.7% for wild-type mice and 69.5% for tumor-bearing mice on day 7).In vivo dissociation of 177 Lu-DOTA-ATPS mAb can decrease therapeutic efficacy and increases bone marrow toxicity.Despite maintaining high in vitro stability (85.5% at 37 • C on day 7) in serum, 177 Lu-DOTA-ATPS mAb exhibited significant bone marrow uptake of free 177 Lu (27.0% for wild-type mice and 39.3% for tumor-bearing mice on day 7).This can be explained by two factors: first, the presence of various blood proteins like transferrin and albumin that strongly bind to 177 Lu and, furthermore, the dilution of 177 Lu-DOTA-ATPS mAb in vivo [28].Overcoming this limitation is crucial, as researchers are actively developing new chelators [31].Further studies to improve the in vivo stability of 177 Lu-DOTA-ATPS mAb are required.
ATPS is normally located in the inner mitochondrial membranes as part of the mitochondrial respiratory complex.It participates in ATP production using a proton gradient generated by mitochondrial respiratory complex I-IV [32].Interestingly, ATPS can also be found on the surface of some cancer and endothelial cells, known as ectopic ATPS.This ectopic ATPS can be a binding site for angiostatin [3].Therefore, ATPS could serve as a novel target for anti-angiogenic cancer therapies.As previously demonstrated, the anti-ATPS mAb used in this study can target tumor vasculature and cancer cells [9].In this study, 177 Lu-DOTA-ATPS mAb showed a significant inhibitory effect on MKN-45 gastric cancers.TGI of 177 Lu-DOTA-ATPS mAb (82.8%) was greater than that of unlabeled ATPS mAb (46.6%).Additionally, immunohistochemistry with an anti-CD31 antibody revealed minimal staining in tumors treated with 177 Lu-DOTA-ATPS mAb (Figure 7B).Similarly, minimal anti-CD31 staining was observed in tumors treated with either single-agent sunitinib or combination therapy (Figure 8B).These findings indicate that the anticancer efficacy of these therapeutic strategies is likely mediated through targeting tumor angiogenesis.
Combination therapy offers a significant advantage by enhancing anticancer effects while potentially reducing side effects compared to single-agent treatment.In this study, combination therapy demonstrated a synergistic increase in therapeutic efficacy (TGI = 70.3%)compared to sunitinib alone (37.8%).
Furthermore, 177 Lu-DOTA-ATPS mAb alone (TGI = 54.6%) also demonstrated a greater therapeutic effect than sunitinib alone.This finding suggests promising potential for the clinical application of 177 Lu-DOTA-ATPS mAb in the future.Chemotherapeutic agents, such as tyrosine kinase inhibitors, have been known to show a broad spectrum of adverse effects in both the hematologic system and nonhematologic systems [33]. 177Lu-based radiopharmaceutical therapies have also been associated with various side effects, some of which are serious hematologic diseases [34,35].Such side effects could obstruct the appropriate treatment of cancer patients, leading to a reduction in dosage or discontinuation of therapeutic agents.Based on the results of this study, combining 177 Lu-radioimmunotherapy with conventional chemotherapy could decrease the therapeutic doses of each treatment, leading to fewer adverse effects than single-agent therapy.However, further clinical studies should be conducted. 177Lu decays by emitting two γ-rays (208 and 113 KeV), which are ideal for gamma camera imaging.While we employed 18 F-FDG PET for tumor visualization in this study, whole-body gamma camera imaging could directly visualize or predict the biodistribution of 177 Lu-DOTA-ATPS mAb.A limitation of this study is the lack of a small animal gamma camera, which prevented us from performing this complementary imaging modality.
We were unable to achieve tumor-free survival as tumors persistently grew in all groups, whether they were subjected to single or combination treatments.In the combination treatment group, tumors from two mice did not exhibit growth until the fourth week of therapy, although they did not completely disappear.In contrast, all tumors in the other groups showed significant growth by the fourth week of treatment.In this study, we initiated treatments when the tumors reached approximately 200 mm 3 in size.For a more accurate evaluation of tumor-free survival, treatments should ideally be started earlier.This is another limitation of the study.
In our previous study [9], we categorized six types of cancer cells into two groups based on their membranous ATPS expression, as determined by Western blot analysis and immunofluorescence microscope findings.From these, we chose MDA-MB-231 (which has high ATPS expression) and PC-3 (which has low ATPS expression) for further comparison.We evaluated these cells using in vitro cellular uptake, binding, and in vivo tumor uptake with 89 Zr-labeled ATPS mAb and positron emission tomography imaging (utilizing the same mAb as in this study).The MDA-MB-231 cells exhibited significantly higher cellular uptake, binding capability (Kd), and in vivo tumor uptake than the PC-3 cells.Based on these results, we hypothesized that cells demonstrating higher in vitro cellular uptake would inevitably show higher in vivo tumor uptake, leading to increased therapeutic efficacy.While it is a well-established concept in clinical radioimmunotherapy to predict therapeutic outcomes using diagnostic radiopharmaceuticals with the same antibodies, it would be beneficial to evaluate the ATPS expression of cancer cells concurrently with cellular uptake.As an alternative, comparing the therapeutic effects between tumors with high ATPS expression and those with lower ATPS expression could aid in drawing more credible conclusions.This is also acknowledged as a limitation of this study.