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. 2025 Apr 29;15:50. doi: 10.1186/s13550-025-01236-4

Evaluation of a simplified radiolabeling method for a PARP inhibitor in an animal model of breast cancer

Chi-Chang Weng 1,2,6,✉, Chao-Chih Chiang 3, Yi-Hsiu Chung 2, Yi-Pei Ho 1, Yu-Chuan Chang 1,4, Ing-Tsung Hsiao 1,4, Robert H Mach 5
PMCID: PMC12040802  PMID: 40301197

Abstract

Background

Several poly (adenosine diphosphate–ribose) polymerase (PARP) inhibitors were recently approved by the US Food and Drug Administration for use in cancer treatment. To facilitate the discovery of novel PARP-targeting ligands, a radioiodinated ligand, I-125-KX1, was developed and validated for its specificity to PARP-1; however, its preparation procedure is time-consuming. The present study employed a solid-phase extraction (SPE) method in the radiolabeling procedure of I-123/125-KX1 and evaluated its binding specificity by using receptor binding assays, autoradiography, and in vivo single photon emission computed tomography (SPECT) imaging technique.

Results

Through the incorporation of the SPE purification method as the final step in the radioiodination procedure, the resultant product I-123/125-KX1 exhibited high radiochemical purity (> 99%) and an acceptable radiochemical yield (58.6% for I-123-KX1, 73.3% for I-125-KX1). The binding characteristics of this radiotracer were validated through saturation binding assays conducted on MDA-MB-231 and MCF-7 cells. The Kd values obtained for the tracer (~ 1.0 nM) was consistent with values reported in the literature, and the Bmax values of these two cell lines (2017 ± 178 fmol/mg on MDA-MB-231 vs. 1393 ± 105 fmol/mg on MCF-7) were in line with the results from Western blot analyses. To demonstrate the in vivo imaging ability of I-123-KX1 prepared in this study, an MDA-MB-231 tumor animal model was used and the tracer displayed a suitable uptake on the tumor tissues (6.9 ± 0.8%ID/mL). The binding specificity of the SPE-purified I-125-KX1 was further verified using in vitro autoradiography in conjunction with various PARP inhibitors. Additionally, an anti-PARP-1 immunohistochemistry experiment was conducted, which revealed that the autoradiograms of the radiotracer displayed a similar pattern.

Conclusions

This suggests that the I-123/125-KX1 prepared using the SPE method showed some comparable properties to those from the traditional method, indicating its potential suitability for future radioligand preparation in PARP studies. However, further characterization studies may be needed to confirm its efficacy.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13550-025-01236-4.

Keywords: PARP, I-123/125-KX1, SPE, Breast cancer, Saturation binding assay, In vitro autoradiography, In vivo SPECT imaging

Introduction

Breast cancer is recognized as the most common cancer worldwide among women. According to estimates by a report in Cancer Statistics, up to 297,790 women in the United States will receive diagnoses of invasive breast cancer in 2023, and 55,720 women will receive diagnoses of noninvasive (in situ) breast cancer in 2023 [1]. Primary treatment modalities for breast cancer include surgical interventions, radiotherapy, and chemotherapy. These treatment strategies may not eliminate cancer from the body. Surgical interventions, for instance, have inherent limitations that can lead to potential tumor recurrence [2].

Radiotherapy and chemotherapy should be applied cautiously due to their high toxicity [3–7]. Recently, promising approaches for cancer treatment have emerged that specifically target certain receptors, such as the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER2), on cancer cells [8–10]. Substantial success has been achieved with compounds that target these receptors, except in the case of triple-negative breast cancer, which receives limited benefits from such interventions [11–13].

Mutations in BRCA1 and BRCA2 genes have been associated with abnormal proliferation of triple-negative breast cancer cells [14, 15]. BRCA1 and BRCA2 genes are crucial for maintaining DNA repair mechanisms, particularly in response to double-strand breaks in the human genome [16]. Overexpression of the enzyme poly(adenosine diphosphate–ribose) polymerase (PARP) was associated with BRCA1- and BRCA2-related cancers in 2005 [17]. Over the past decade, numerous PARP inhibitors have demonstrated efficacy in targeting cancers harboring BRCA gene mutations [18]. PARP inhibitors have potential as monotherapies and in combination with chemotherapy or radiotherapy [19].

PARP inhibitors are specifically designed for cancers characterized by BRCA gene mutations, particularly those exhibiting PARP enzyme overexpression. To facilitate the identification of cancers with high PARP enzyme expression, researchers have recently proposed various radiotracers capable of detecting PARP-1, one of the most abundant enzymes in the PARP family [20]. Notable examples of these radiotracers include I-125-KX1 and F-18-FTT, which have demonstrated the ability to detect PARP-1 enzyme expression in cancer cells and in small animal studies [21–23]. Although PET (positron emission tomography) tracers provide superior image resolution, challenges such as their short half-life and the requirement for cyclotron production may limit their availability. Moreover, many radioiodinated PARP inhibitors have been identified as potential treatment strategies, particularly those using Auger electrons or beta particles for cancer therapy [24, 25].

The conventional preparation procedure for most radioiodinated PARP inhibitors typically includes a time-consuming semipreparative high-performance liquid chromatography (HPLC) purification step, which may not be convenient for future laboratory personnel. Therefore, this study introduced a simplified preparation method for radioiodinated KX1 by using a C18 solid-phase extraction (SPE) cartridge. The biological characteristics of the radiolabeled compound were evaluated through receptor binding assays across various breast cancer cell lines. Furthermore, KX1 was labeled with I-123 to assess its in vivo imaging capabilities as a PARP-1-specific SPECT radiotracer in MDA-MB-231 breast cancer cell–bearing animal models.

Materials and methods

Radiochemistry

To simplify the preparation procedure for radioiodinated I-123/125-KX1, a tracer purification method by using a C18 SPE cartridge (7020-03, J.T. Baker, Phillipsburg, NJ, USA) was applied. This approach has been previously employed for the radioiodinated Aβ ligand, I-125-IMPY [26]. The modified procedure used in this study is detailed as follows (Fig. 1). First, 10 µg (~ 18 nmole) of the ligand precursor (1-(4-(tributylstannyl)phenyl)-8,9-dihydro-2,7,9a-triazabenzo[cd]azulen-6(7 H)-one) was dissolved with 50 µL of ethanol. Subsequently, 100 µL of Na123I with about 777 MBq (NARI, Taoyuan, Taiwan) or 10 µL of Na125I with around 36 MBq (Izotop, Budapest, Hungary), 100 µL of 3% H2O2, and 50 µL of 4 N HCl were added to the same vial containing the dissolved precursor. The radiolabeling reaction was conducted at room temperature (RT) for 10 min. The reaction was terminated by the addition of 50 µL of NaHSO3, followed by the introduction of 3 mL of a saturated NaHCO3 aqueous solution to neutralize the reaction mixture. The crude product was then loaded onto a C18 SPE cartridge (which was preconditioned with 3 mL of ethanol and 6 mL of ddH2O before use), allowing for the removal of unreacted radioiodine and potential radiochemical impurities by using 10% and 20% ethanol aqueous solutions, respectively. The final product was eluted with 1 mL of 100% ethanol.

Fig. 1.

Fig. 1

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I-123/125-KX1 radiolabeling procedure

The radiochemical purity of I-123/125-KX1 was evaluated using a Waters 1527 Binary HPLC Pump (Waters, Milford, MA, USA) equipped with an analytical C18 column (Gemini C18, 5 μm, 4.6 × 250 mm; Phenomenex, Torrance, CA, USA). The mobile phase was freshly prepared from a mixture of acetonitrile and 0.1 M ammonium formate (pH 4.5) in a 4:6 (v/v) ratio, with the flow rate set at 1 mL per minute for sample analysis. Ultraviolet absorbance was monitored at 254 nm for the KX1 standard (1-(4-iodophenyl)-8,9-dihydro-2,7,9a-triazabenzo[cd]azulen-6(7 H)-one) by using a UV2489 ultraviolet and visible light detector (Waters), and radioactivity was measured using a B-FC-1000 flow count detector (Bioscan, Washington, DC, USA). Meanwhile, the precursor for KX1 and its standard were generously provided by Dr. Mach and synthesized using a previously published method [23].

Cell culture

Two breast cancer cell lines—MDA-MB-231 and MCF-7—were obtained from the Bioresource Collection and Research Center (#60425 for MDA-MB-231, #60436 for MCF-7, Hsinchu, Taiwan). MDA-MB-231 cells were cultured in minimum essential medium (41500-034, Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (26140079, Gibco) and 1% penicillin/streptomycin (15140-122, Gibco) at 37 °C with 5% CO2. MCF-7 cells were cultured in DMEM (12100-046, Gibco), supplemented with 10% fetal bovine serum (Gibco), 1% penicillin/streptomycin (Gibco), and 1.5 g/L sodium bicarbonate (pH 7.1–7.2; S5761-1KG, Sigma, St Louis, MO, USA).

Western blotting

The experimental procedures were primarily conducted in accordance with the manufacturer’s recommended protocols. First, MDA-MB-231 and MCF-7 cells were lysed using radioimmunoprecipitation assay buffer (TAAR-ZBZ5, TOOLS, Taiwan) supplemented with a protease inhibitor (TAAR-BBI2, TOOLS). After centrifugation, the supernatant was collected (1 × 107 cells/100 µL) and maintained on ice. The samples were then mixed with a loading buffer (TAAR-TB2, TOOLS) and boiled at 95 °C for 5 min. After the mixture was cooled to RT, different cell samples were loaded into a precast gel (QP4510, SMOBIO, Taiwan) and subjected to electrophoresis at 150 V for 55 min. After electrophoresis, the gel was removed, and the proteins were transferred onto an activated polyvinylidene difluoride (PVDF) membrane (Cat#: BSP0161, PALL, USA). The transfer procedure was conducted using a transfer buffer at 100 V for 70 min. Following transfer, the membrane was removed from the chamber and incubated in a blocking solution of 5% milk at RT for 1 h. Subsequently, the PVDF membrane was incubated with primary antibodies PARP-1 (diluted 1:1000, #9542, Cell signaling) and GAPDH (diluted 1:32000, #C06012, Croyex) overnight at 4 °C.

Following incubation, the PVDF membrane was washed with phosphate-buffered saline with 0.5% Tween 20 for 5 min, thrice. The PVDF membrane was then incubated with secondary antibodies, specifically goat anti-mouse IgG (RA-BZ102, TOOLS) diluted at 1:15,000 and goat antirabbit IgG (RA-BZ202, TOOLS) diluted at 1:30,000 at RT for 1 h. After the incubation, the membrane was treated with enhanced chemiluminescence reagent (TU-ECL02, TOOLS) and scanned using a two-dimensional gel imaging system (Amersham Imager 600, GE, USA) to detect chemiluminescent signals. The resultant images were analyzed using ImageJ software, following a method published elsewhere [27].

Cell membrane Preparation

To validate the binding properties of the radioligand prepared using the simplified protocol, MDA-MB-231 and MCF-7 cells were used for the saturation receptor binding assay. The cell membrane harvesting procedure was conducted in accordance with published methods [28]. Briefly, both cell lines were scraped from culture dishes by using ice-cold phosphate-buffered saline (PBS) and collected through centrifugation at 1000 rpm and 4 °C for 10min. The resultant cell pellets were immediately stored at − 80 °C until further use. For the preparation of cell membrane homogenates, the cell pellets were resuspended in 10 mL of ice-cold PBS and homogenized using a Hei-TORQUE Core Overhead Stirrer (Heidolph, Schwabach, Germany) at a speed setting of 2 for 30 s. The homogenates were then centrifuged for 20 min at 31,000×g at 4 °C. After centrifugation, the supernatant was discarded, and the pellets were resuspended in 1 mL of ice-cold PBS before being stored at − 80 °C in a freezer until use.

Saturation binding assay

The protocols for the I-125-KX1 binding assays were conducted following established references with a minor modification [29]. Briefly, 20–30 µg of MDA-MB-231 or MCF-7 cell membrane protein was incubated with different concentrations of I-125-KX1, prepared in a range from 1 nM to 1 µM, for 60 min at 37 °C. The concentration of the radioligand was calculated according to the steps published before [30]. Nonspecific binding was assessed using 20 µM olaparib (HY-10162, MCE, USA). After incubation, the bound ligands were filtered using an M-24 Brandel filtration system (Brandel, Gaithersburg, MD, USA), and the samples were collected on glass fiber filter papers (Whatman grade 934-AH, GE Healthcare Bio-Sciences, USA). Radioactivity was then quantified using a Hidex AMG Automatic Gamma Counter (Turku, Finland). All dissociation constant (Kd) and maximum binding (Bmax) values were calculated using nonlinear regression analysis with GraphPad Prism 6 (GraphPad, Boston, MA, USA). Protein concentrations were determined using the method described by Lowry et al. [31], with bovine serum albumin as a standard.

Tumor animal model Preparation

First, MDA-MB-231 cells (5 × 107 cells/mL) were prepared in PBS supplemented with 10% basement membrane matrix (#354234, Corning Matrigel). Subsequently, a volume of 100 µL containing 5 × 106 cells was subcutaneously inoculated into the right ventral flank of five male Balb/c nude mice (20–25 g; National Laboratory Animal Center, Taiwan). All procedures involving animal care and experimentation were approved by the Institutional Animal Care and Use Committee of Chang Gung University.

In vitro autoradiography

The tumor-bearing mice were sacrificed, and their tumors were rapidly excised and embedded in an optimal cutting temperature medium (Sakura Finetek, USA). Tumor sections were prepared at a thickness of 30 μm by using a Leica CM3050S Cryostat and stored at − 80 °C for subsequent in vitro autoradiography and immunohistochemistry studies.

Selected sections were categorized into vehicle and blocking groups in preparation for autoradiographic experiments. All tumor sections were incubated with 0.4 nM I-125-KX1 for 1 h at RT. Concurrently, the sections of the blocking group were incubated with different PARP inhibitors (olaparib, Veliparib, AG-14361, and KX1 standard), prepared with 40 µM (the first three compounds were purchased from MCE, USA; the KX1 standard was a gift from Dr. Mach), and dissolved in 50 mM Tris buffer. After three 3-min washes with ice-cold Tris buffer (50 mM), the sections were briefly rinsed with distilled water for 30 s. After drying, the radiolabeled sections were exposed to a BAS-SR 2040 imaging plate (20 × 40 cm, Fujifilm, Japan) for 24 h and subsequently scanned using a Typhoon 5 image reader (Cytiva, USA). The regions of interest were delineated and analyzed using Multi Gauge v3.0 software (Fujifilm, Tokyo, Japan) to quantify tracer uptake in the different radiolabeled sections.

Immunohistochemistry

Adjacent sections from the autoradiographic studies were first fixed with ice-cold acetone in RT for 5 min. This was followed by blocking the endogenous hydrogen peroxidase activity by using a commercialized H2O2 inhibitor and a protein blocker for 10 min. After three 5-min washes with ice-cold PBS, these slides were incubated with an anti-PARP-1 antibody (#9532, Cell signaling, USA) overnight at 4 °C and then incubated with a goat antirabbit HRP-conjugated secondary antibody for 1 h at RT. Subsequently, the PARP-1 signal on the slides was detected using a DAB (ABC) Detection immunohistochemistry (IHC) Kit (ab64261, Abcam, USA). After mounting with coverslips, the stained images were captured using a ZEISS Axio Imager M2 microscope (ZEISS, USA).

In vivo I-123-KX-1 SPECT/CT image acquisition

To further evaluate the in vivo imaging capability of the I-123-KX1, mice bearing MDA-MB-231 underwent SPECT/CT imaging. Briefly, images were acquired using a NanoSPECT/CT scanner (Mediso, Budapest, Hungary). Approximately 0.4–0.6 MBq of I-123-KX1 was administered intravenously via the tail vein. Static SPECT images were collected for 30 min, with the animals maintained under 2% isoflurane anesthesia 1 h after tracer injection. Regions of interest, including the tumor and muscle, were manually delineated, and the uptake values were calculated as percentage of injected dose per mL (%ID/mL). All images were analyzed using PMOD version 3.7 (PMOD Technologies LLC, Fällanden, Switzerland).

Statistics.

All data were analyzed using GraphPad Prism 6 software. The results are presented as the mean ± standard error of the mean. Differences among groups were assessed using a two-tailed Student’s t test. P < 0.05 was considered statistically significant.

Results

Radiochemistry

The radioiodination of I-123/125-KX1 by using a C18 SPE effectively reduced the overall labeling procedure to approximately 30 min. The radiochemical yields (after purification) were 58.6% ± 16.9% for I-123-KX1 and 73.3% ± 15.3% for I-125-KX1. Detailed activity distribution data throughout the radiolabeling process are provided in Table S1. The final SPE-purified product exhibited high radiochemical purity (> 99%), as confirmed using HPLC analysis. A representative chromatogram is presented in Fig. 2. Moreover, the molar activity was 85.0 ± 28.6 GBq/µmole (n = 3) for I-125-KX1 and 211.0 ± 110.6 GBq/µmole (n = 3) for I-123-KX1 prepared in this study.

Fig. 2.

Fig. 2

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Representative HPLC chromatogram for (A) UV chromatogram of I-125-KX1; (B) Radio chromatogram of I-125-KX1; (C) authentic KX1 standard chromatogram

Western blots

PARP-1 expression levels in MDA-MB-231 and MCF-7 cells were evaluated through Western blot analysis, with representative images displayed in Fig. 3. The bands corresponding to anti-PARP-1 or anti-GAPDH are presented in Fig. 3A, and their image quantification results are presented in Fig. 3B. The histograms indicate that PARP-1 expression in MDA-MB-231 cells was 1.84-fold higher than that in MCF-7 cells.

Fig. 3.

Fig. 3

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(A) Representative Western blotting images illustrating PARP-1 expression levels in MCF-7 and MDA-MB-231 cells. (B) Quantification of Western blot images. ****p < 0.001

Saturation binding assay

The binding characteristics of I-125-KX1 on MDA-MB-231 and MCF-7 cell membranes are displayed in Fig. 4. The radiotracer demonstrated favorable and comparable binding affinity to PARP-1 on both cell membranes (Kd = 1.4 ± 0.3 nM on MDA-MB-231 vs. 1.0 ± 0.2 nM on MCF-7). Moreover, the maximum binding capacity (Bmax) value was higher in MDA-MB-231 cell membranes (2017 ± 178 fmol/mg) than in MCF-7 cell membranes (1393 ± 105 fmol/mg), as shown in Fig. 4B. The Bmax ratio between these cell lines was 1.45, closely aligning with the Western blot results. These findings further confirm that the simplified preparation method for I-125-KX1 effectively preserves its binding properties.

Fig. 4.

Fig. 4

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(A) Saturation binding curves of I-125-KX1 on MDA-MB-231 and MCF-7 cell membranes. (B) Bmax values. *p < 0.05

In vivo I-123-KX1 SPECT imaging study

Representative SPECT images of I-123-KX1 in an MDA-MB-231 tumor-bearing animal model is presented in Fig. 5. The in vivo SPECT data demonstrated a notably higher accumulation of radioactivity within the tumor tissue compared with the surrounding muscle, confirming the tracer’s specificity for PARP-1. To further evaluate the differences in tracer uptake across regions, quantification results are presented in Fig. 6. The tracer uptake in the MDA-MB-231 tumor region reached 6.9 ± 0.8%ID/mL, in contrast to 3.1 ± 0.4%ID/mL in adjacent muscle tissue. The tumor-to-muscle uptake ratio of 2.3 indicates a potential for I-123-KX1 in monitoring PARP-1 expression in vivo, suggesting its suitability as a candidate for evaluating PARP-1 tumor progression in future studies.

Fig. 5.

Fig. 5

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In vivo SPECT/CT images of I-123-KX-1 in MDA-MB-231 tumor-bearing mice (n = 3). Location of tumor inoculation is indicated by red circles

Fig. 6.

Fig. 6

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Quantification results of SPECT imaging in MDA-MB-231 tumor-bearing mice (n = 3). (A) Tracer uptake in various tissues. (B) Summary of tumor-to-muscle ratio. *p < 0.05

In vitro autoradiography and IHC

To verify the specific binding characteristics of I-125-KX1 synthesized using the simplified labeling procedure, MDA-MB-231 tumor tissue sections from the tumor-bearing mouse were analyzed through in vitro autoradiography. The autoradiograms, as presented in Fig. 7, indicated that the binding I-125-KX1 was efficiently blocked by various PARP inhibitors, such as olaparib, Veliparib, AG-14,361, and KX1 standard. This outcome confirms the high specificity of I-125-KX1 for PARP-1. Notably, these autoradiographic findings are consistent with the anti-PARP-1 IHC staining patterns, suggesting strong alignment in localization of PARP-1 expression between the two methods. To further confirm the specific blocking ability of each PARP inhibitor, the autoradiographic images were analyzed for image density, and the results are presented in Figure S1.

Fig. 7.

Fig. 7

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Representative images of PARP immunohistochemistry and in vitro autoradiography of I-125-KX1 competed with different PARP inhibitors on MDA-MB-231 tumor tissue sections. The autoradiograms clearly demonstrate the specific binding of I-125-KX1, prepared using this simplified method, which is effectively blocked by PARP-1 selective compounds

Discussion

PARP inhibitors, such as olaparib, have demonstrated efficacy in cancer treatment, leading to their clinical approval on December 19, 2014 [32]. However, the therapeutic success of PARP inhibitors in oncology is largely contingent upon the expression levels of PARP enzymes within tumor tissues [33]. To improve patient selection for PARP inhibitor therapy, different radiotracers have been developed to facilitate the diagnostic assessment of PARP expression. Among these, a radioiodinated rucaparib derivative, KX1, has been reported to effectively detect PARP-1 [34]. In published studies on saturation binding, KX1 labeled with I-125 exhibited minimal binding to MEF PARP1 KO−/− cells, confirming its specificity for PARP-1. Given its demonstrated affinity for PARP-1, I-125-KX1 has been further explored for potential therapeutic applications across different tumor cells or tumor-bearing animal models [35–37].

Although the results obtained using I-125-KX1 appear promising, the complexity and time-intensive nature of its preparation may limit its accessibility. Another study described a labeling procedure that involves incubating the reaction mixture at 100 °C for 30 min, followed by purification by using an HPLC system equipped with a semipreparative C18 column. Even after HPLC purification, the collected crude product still requires further elution with a C18 SPE cartridge [34]. To simplify the labeling process and enhance the availability of radioiodinated KX1 for broader research applications, we adapted a methodology inspired by a 2004 study on a beta-amyloid-specific radioligand, I-125-IMPY. That study introduced a streamlined approach using a C4 SPE cartridge for purification, achieving high radiochemical yield (> 50%) and purity (> 95%) [26]. Following the principles outlined by Kung et al. in 2004, we evaluated and provided a more convenient preparation protocol for I-125-KX1 by using C18 SPE purification, reducing labeling time to under 30 min. The I-123/125-KX1 prepared in this study achieved a radiochemical yield of 58.64% and 73.29%, respectively, which is comparable to previously reported values (60–70% for both radioligands) [37]. Quality control analysis using HPLC confirmed a radiochemical purity of > 99%. Additionally, the molar activity and radiochemical yield of I-125-KX1 (85.0 GBq/µmol) closely matched previously reported values (81.4 GBq/µmol) [36]. To the best of our knowledge, no prior data on the molar activity of I-123-KX1 has been reported. However, our findings (211.0 GBq/µmol) suggest that its molar activity is comparable to that of I-123-PARPi-01, as previously documented (279 GBq/µmol) [38].

To assess whether the radioiodinated KX1 retains bioactivity toward the PARP-1 enzyme, a saturation binding assay was conducted using I-125-KX1 on membrane preparations from two breast cancer cell lines. The binding affinity (Kd) obtained using this simplified preparation method from the was comparable to reported values (~ 1.4 nM vs. 5.6 nM). Notably, the maximum binding capacity (Bmax value) observed in this study on the MDA-MB-231 group was comparable to the published results from assays conducted on intact cells before (2017 ± 178 vs. 2867 ± 78) [34]. This suggests that assays using cell membranes might yield comparable results to those using intact cells. Moreover, the Bmax values for MDA-MB-231 and MCF-7 cell membranes revealed significant differences, consistent with the findings from the Western blot analysis, which reflect variations in PARP-1 expression between these two cell lines [39]. This result underscores the use of I-125-KX1 in quantifying differential PARP-1 enzyme expression.

To evaluate the potential of this simplified preparation method for other radioiodine, such as I-123 for future in vivo SPECT imaging studies, we also prepared I-123-KX1 by using this protocol. The radiochemical yield for I-123-KX1 was slightly lower than that of I-125-KX1 (58.64% for I-123-KX1 vs. 73.29% for I-125-KX1), possibly due to the larger reaction volume required when using Na123I. However, the in vivo preliminary results of SPECT indicate that I-123-KX1 is capable of differentiating tumor tissue in the MDA-MB-231 tumor-bearing mice, achieving a tumor-to-muscle ratio of approximately 2.3. Comparable effort on the same tumor model was made by Sankaranarayanan et al. by using an olaparib-based tracer, I-123-PARP inhibitor-01. Unfortunately, their findings revealed a limited tumor uptake 24 h after tracer injection [38]. Moreover, Riad et al. also applied the same radiotracer (I-123-KX1) but purified with a semipreparative HPLC system on an ovarian cancer animal model. However, the tumor-to-muscle ratio is only 1.29 [37], suggesting that maybe the simplified radiotracer preparation method present herein could be a more efficient way for SPECT imaging studies.

To evaluate the specific binding characteristics of I-125-KX1 prepared in this study, in vitro autoradiography blocking experiments with different PARP inhibitors were conducted, along with IHC staining by using a PARP-1 specific antibody. These procedures were applied to a series of tumor tissue sections harvested from an MDA-MB-231 tumor-bearing mouse. The autoradiograms revealed a clear and distinct binding pattern, indicating that the I-125-KX1 prepared using the simplified protocol exhibited strict binding to PARP-1 specific sites. This binding could be effectively blocked by other highly PARP-1-specific ligands, such as olaparib, veliparib, AG-14,361, and its standard. Furthermore, the binding pattern observed was in line with the findings from the IHC images, corroborating findings from published data [22].

Conclusions

The comprehensive approach used in this study—encompassing radiochemistry, Western blotting, saturation binding assays, autoradiography, and IHC—highlights the potential of I-125-KX1 as a valuable tool for investigating PARP-1 expression in breast cancer. Moreover, the observed differences in PARP-1 expression between MDA-MB-231 and MCF-7 cells underscore the need for personalized treatment strategies that specifically target PARP-1 in breast cancer therapy. These findings pave the way for further exploration of PARP inhibitors in breast cancer treatment, with the potential to enhance the precision and effectiveness of therapy in specific subtypes of the disease.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1 (193KB, docx)
Supplementary Material 2 (23.3KB, docx)

Acknowledgements

Authors thank Laboratory Animal Center, Chang Gung Memorial Hospital, Linkou, for molecular imaging and technical support, and the Microscopy Center at Chang Gung University for technical assistance. We also would like to thank all the anonymous reviewers and the editor for their comments.

Abbreviations

PARP

Poly (adenosine diphosphate–ribose) polymerase

SPE

Solid-phase extraction

SPECT

Single photon emission computed tomography

Bmax

Maximum binding capacity

HER2

Human epidermal growth factor receptor 2

BRCA1/2

Breast cancer gene 1/2

DNA

Deoxyribonucleic acid

PET

Positron emission tomography

HPLC

High-performance liquid chromatography

NARI

National atomic research institute

RT

Room temperature

PVDF

Polyvinylidene difluoride

GAPDH

Glyceraldehyde 3-phosphate dehydrogenase

PBS

Phosphate-buffered saline

Kd

Dissociation constant

IACUC

Institutional animal care and use committee

IHC

Immunohistochemistry

DAB

3,3’-Diaminobenzidine

ABC

Avidin-biotin complex

CT

Computed tomography

%ID/mL

Percentage injected dose per mL

Author contributions

C.C.C., Y.H.C., and R.H.M. supervised and administered the project. C.C.W. contributed to the study conception and design. C.C.W. conducted radiosyntheses. C.C.C. and Y.C.C. carried out the cell culture and experiments with cell lines. C.C.C., Y.H.C., and Y.P.H. handled the care, treatment and preclinical molecular imaging examination of the animals. Y.P.H. conducted cryosections and staining. C.C.C. and Y.H.C. analyzed the data and performed statistical analyses. C.C.W., Y.C.C., I.T.H., and R.H.M. provided material and resources. C.C.W., C.C.C., and Y.H.C. interpreted the analyzed data. C.C.W. wrote the manuscript. Y.C.C., Y.H.C., and R.H.M. corrected the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Science and Technology Council, Taiwan (Grant MOST 108-2314-B-182-063- for CCW), the grants from the Research Fund of Chang Gung Memorial Hospital (CMRPG3N0461 and CMRPG3N0462 for YHC, CMRPD1M0242 for CCW), grant from the National Atomic Research Institute of Taiwan (Grant NSC-112-80-01-03-01-02 for CCW), and the grant from the Healthy Aging Research Center, Chang Gung University, Taoyuan, Taiwan (URRPD1P0201).

Data availability

The analyses of the data supporting the conclusions of this article is included within the article and the supplementary file. The raw datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

All procedures involving animal care and experimentation were approved by the IACUC of Chang Gung University (IACUC number: CGU108-050).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (193KB, docx)
Supplementary Material 2 (23.3KB, docx)

Data Availability Statement

The analyses of the data supporting the conclusions of this article is included within the article and the supplementary file. The raw datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

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