Combined use of niraparib enhanced the inhibitory effect of Anti-GD2 antibody on osteosarcoma cells

Purpose This study aims to investigate the effect of Niraparib in combination with an Anti-GD2 Antibody on osteosarcoma cells. Methods Scratch test was utilized to assess cell migration capacity, while the Transwell experiment was utilized to evaluate cell invasion potential. Cell proliferation was measured using the CCK8 experiment. The affinity between the anti-GD2 antibody and its antigen was determined via ELISA. Tumor growth was evaluated through animal experiments. Western blotting, QRT-PCR, and histological analysis were conducted to examine the expression of relevant proteins and mRNAs. Results MG63 cell line was used for an example. The scratch test showed that the migration rate of osteosarcoma cells in Niraparib + Anti-GD2 group was 1.07 ± 0.04 after 48 h, and 0.34 ± 0.04 in the Control group. Transwell experiment showed that the invasion ability of osteosarcoma cells in Niraparib + Anti-GD2 group was 21.0 ± 1.5, and that in Control group was 87.7 ± 2.9. CCK8 experiment showed that the absorbance value of Niraparib + Anti-GD2 group was 0.16 ± 0.10 on day 5, and that of the Control group was 0.76 ± 0.09. Western blotting showed that the expression levels of BALP and CICP in Niraparib + Anti-GD2 group were 0.751 ± 0.135 and 1.086 ± 0.115, respectively, and those in Control group were 1.025 ± 0.143 and 1.216 ± 0.168, respectively. QRT-PCR results showed that the absorbance values of Niraparib + Anti-GD2 group were 0.173 ± 0.065 and 0.170 ± 0.078 on day 14. The results of animal experiments showed that on day 5, the tumor volume of the Control group was 2433 ± 391, and that of the Niraparib + Anti-GD2 group was 1137 ± 148. Histological analysis showed that the mean density values of Niraparib + Anti-GD2 group were 0.19 ± 0.08 and 0.22 ± 0.07, and those of Control group were 0.26 ± 0.09 and 0.29 ± 0.10. Conclusion The combination of Niraparib and Anti-GD2 antibody significantly inhibits Osteosarcoma cells. Supplementary Information The online version contains supplementary material available at 10.1007/s12672-024-01166-y.


Introduction
Osteosarcoma (OS) is the most common primary bone malignancy, accounting for approximately 35% of all orthopedic tumors [1].OS is a serious and destructive disease characterized by high local aggressiveness and a tendency to metastasize to the lungs and distant bones.The cure rate for OS is low, which further decreases if there are metastases.

Animal selection
In the present study, BALB/cA Nu/Nu nude mice were selected.The nude mice were 4 to 6 weeks old and weighed between 15 and 20 g. Specific pathogen-free (SPF) conditions were maintained to minimize the risk of infection.All animal experimental protocols conducted within this study have been rigorously reviewed and approved by the Ethical Review Committee of Renhe Hospital, China Three Gorges University.Our research is conducted in strict accordance with the ethical principles outlined in the Basel Declaration and adheres to all pertinent national laws and policies.We hereby confirm that in the course of our study, conducted under the auspices of the Ethical Review Committee (ERC) of Renhe Hospital, China Three Gorges University, the maximum tumor size/burden guidelines as established by the ERC have been strictly adhered to.No animal involved in our research has exceeded the maximum tumor size/burden limit of 1.5 cm in diameter or 10% of body weight, as approved by the ERC.

Cells lines and reagents
SYBR Green Real-Time PCR Master Mix (Xavier Biotechnology Co., LTD.), bone alkaline phosphatase (BALP), type I collagen carboxyl terminal propeptide (CICP), and GAPDH antibodies were purchased from Xavier Biotechnology Co., LTD.

Trasnswell experiment
On the day before the experiment, a tube of Matrigel Basement Membrane Matrix that had been divided was placed in the refrigerator at 4 ℃ overnight, previously stored at -20 ℃.The Matrigel Basement Membrane Matrix was diluted at a ratio of 1:8 and coated on the upper surface of the bottom membrane in the Transwell chamber.The residual liquid from the culture plate was removed, and each well was supplemented with 50 μL of serum-free medium containing 10 g/L BSA, incubated at 37 ℃ for 30 min.The cells were digested using conventional trypsin and washed with PBS one to two times to remove any effects from serum.Then, they were resuspended in serum-free medium and adjusted to a cell density of 5 × 105 cells/mL.Next, 200 μL of cell suspension was added to the upper chamber of Transwell chamber while adding 600 μL medium containing 10% FBS to the lower chamber of a 24-well culture plate.The culture plate was placed in a CO2 incubator at 37 ℃ for 24 h.Afterward, cells were taken out, washed twice with PBS, and carefully wiped off from the upper layer of microporous membrane using cotton swabs.Subsequently, cells were fixed with a solution containing 4% paraformaldehyde in a separate well plate for about twenty minutes followed by staining them with crystal violet solution for fifteen minutes.Finally, photographs were captured under an inverted microscope where ten random fields were counted per sample and their average value used for statistical analysis.

CCK8 experiment.
A cell suspension was inoculated into a 96-well plate (100 μL/well).The culture plate was then placed in an incubator for pre-culture for a specific period of time (37 ℃, 5% CO2).Next, 10 μL of CCK8 solution was added to each well.The culture plate was incubated in the incubator for 2 h.Finally, the absorbance at 450 nm was measured using a microplate reader.

Enzyme-linked immunosorbent assay (ELISA)
The GD2 antigen was immobilized in the wells of an ELISA plate and treated with a blocking solution.Then, high-affinity antibodies (positive control), low-affinity or non-specific antibodies (negative control), and experimental group antibodies (GD2 antibody) were properly diluted and added to the wells.After incubation, thorough washing of the wells was conducted.Enzyme-labeled secondary antibodies were subsequently introduced, followed by color development and reaction termination.Finally, the optical density (OD) value of each well was measured to determine antibody affinity.

Western blotting
Protein (40 µg) was extracted, separated by 10% SDS-PAGE, and transferred to polyvinylidene difluoride membranes.The membranes were blocked with 5% skimmed milk, followed by overnight incubation with primary antibodies at 4 °C.Subsequently, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 2 h.Blots were developed using the ECL system.The antibodies used in our study included Anti-BALP antibody and Anti-CICP antibody.

Real-time quantitative reverse transcription
Total RNA was extracted using the TRIzol method, and cDNA was synthesized with the RevertAidTM first-strand cDNA synthesis kit.Real-time polymerase chain reaction (PCR) was performed using the ABI PRISM 7900HT sequence detection system.The expression of osteogenesis-related genes was normalized to GAPDH using the 2−ΔΔCT method.The primer sequences are listed in Table 1.

The establishment of the subcutaneous osteosarcoma model
The BALB/cA Nu/Nu nude mice were selected and randomly allocated into four groups: the Control group, Niraparib group, Anti-GD2 group, and Niraparib + Anti-GD2 combination therapy group.The osteosarcoma cell line (MG63) was prepared as a cell suspension and adjusted to an appropriate concentration before subcutaneously implantation in the nude mice to induce tumor formation.Tumor volume and health status were regularly assessed.Administration of the treatment was performed at a dose of 25 mg/kg once daily.The control group received an equivalent volume of placebo solution.The Niraparib group received Niraparib drug treatment.The Anti-GD2 group received Anti-GD2 antibodies for treatment.The Niraparib + Anti-GD2 combination therapy group received both Nirparab and Anti-GD-antbodies for combined treatment.Tumor growth data were periodically collected throughout the experiment duration of 5 days, after which the experiment was terminated to compare tumor growth among different groups, evaluate drug efficacy, perform statistical analysis, and determine significant differences.

Histological analysis
Mouse tumors were sectioned and rinsed three times with PBS for 1 min each time.Cells were fixed with 4% paraformaldehyde for 15 min at room temperature, then air dried for 5 min and washed three times with PBS for 3 min each time.Cells were permeabilized with 0.5% Triton X-100 for 20 min at room temperature.Cells were then rinsed twice with PBS for 5 min each.Add 3% H2O2 solution in deionized water and incubate for 10 min at room temperature, then rinse with PBS.Add normal goat serum and incubate at 37 °C for 15 min without rinsing.Add primary antibody working solution and incubate overnight at 4 °C, then rinse twice with PBS for 5 min each time.Add HRP-conjugated secondary antibody working solution and incubate at 37 °C for 45 min, then rinse twice with PBS for 5 min each time.DAB substrate was added and the results were visualized under a microscope.Safranin was used as a stain, followed by tissue dehydration.Neutral resin was sealed, observed and photographed using a light microscope.

Statistical analysis
The image processing was performed using Image J. Statistical analyses were conducted using SPSS for Windows 10.All data values were expressed as means ± SD.Comparisons of means among multiple groups were carried out using one-way ANOVA, followed by post hoc pairwise comparisons using Tukey's tests.A two-tailed p-value less than 0.05 was considered statistically significant in this study.

Scratch test
The scratch width did not show a significant difference between the four groups of the two cell lines at 0 h.In the MG63 cell line, the Control group's scratch width almost disappeared at 48 h, with a distance of 0.34 ± 0.04.However, in the Niraparib + Anti-GD2 group, the scratch width remained relatively wide with a distance of 1.07 ± 0.04 (p < 0.05), and this difference was statistically significant.The U2OS cell line exhibited a similar trend as well.These results indicate that combining Niraparib with Anti-GD2 antibody has a significant inhibitory effect on OS cells' migration (Fig. 1).Supplementary Table 1 presented the corresponding statistical data.

Trasnswell experiment
Transwell experiment results showed that in the MG63 cell line, compared with the Control group, the invasive ability of OS cells was inhibited in the Niraparib, Anti-GD2, and Niraparib + Anti-GD2 groups.However, combining Niraparib with Anti-GD2 antibody had the most significant inhibitory effect on the invasion ability of OS cells (Fig. 2).By counting, the number of cells that invaded through the Matrigel Basement Membrane Matrix in the Control group was 87.7 ± 2.9, while it was 21 ± 1.5 in the Niraparib + Anti-GD2 group (p < 0.05), indicating a statistically significant difference.The U2OS cell line exhibited a similar trend.These results demonstrate that combining Niraparib with Anti-GD2 antibody has a significant inhibitory effect on the invasive ability of OS cells.Supplementary Table 2 presented statistical data.

CCK8 assay
The CCK8 assay results showed that the Niraparib group and Anti-GD2 group did not have a significant inhibitory effect on OS cells' proliferation compared to the Control group.However, when Niraparib was combined with Anti-GD2 antibody, it significantly inhibited the proliferation of OS cells (Fig. 3).As culture time increased, the inhibitory effect of Niraparib combined with Anti-GD2 antibody on OS cells' proliferation gradually increased.On day 5, cell proliferation in the Control group was assessed by absorbance as 0.76 ± 0.09 while in the Niraparib + Anti-GD2 group it was 0.16 ± 0.10 (p < 0.05), and this difference was statistically significant.These results indicate that Niraparib combined with Anti-GD2 antibody has a significant inhibitory effect on OS cells' proliferation.Supplementary Table 3 presented the corresponding statistical data.

ELISA
The MG63 cell line was used as an example.After quantifying affinity based on the relative change in OD values, we observed that the positive OD value was 1.300, the negative OD value was 0.125, and the experimental group exhibited an OD value of 1.067 (Table 2).A higher OD value indicates a greater concentration of the target substance and thus signifies stronger affinity.The high-affinity antibody demonstrated significantly elevated OD values compared to its lowaffinity counterpart, highlighting its superior binding capability.By comparing the ratio of OD values, we estimated that the anti-GD2 antibody possesses approximately 86.7% of the affinity displayed by the high-affinity antibody, thereby demonstrating its relatively robust binding capacity.This experimental finding effectively elucidates why anti-GD2 antibodies can still exert their functionality despite lower GD2 levels in MG63 and U2OS cells.

Western blotting
Western blotting results showed that in the MG63 cell line, compared with the Control group, the protein expression levels of BALP and CICP were inhibited in OS cells treated with Niraparib, Anti-GD2 antibody, and Niraparib + Anti-GD2 antibody.However, the combination of Niraparib and Anti-GD2 antibody had the most significant inhibitory effect on the expression levels of BALP and CICP proteins in OS cells (Fig. 4).Through semi-quantitative analysis, the expression levels of BALP and CICP in the Control group were 1.025 ± 0.143 and 1.216 ± 0.168 respectively, while those in the Niraparib + Anti-GD2 group were 0.751 ± 0.135 and 1.086 ± 0.115 respectively (both p < 0.05), (2024) 15:304 | https://doi.org/10.1007/s12672-024-01166-yResearch indicating statistically significant differences between groups.These results demonstrate that Niraparib combined with Anti-GD2 antibody has a significant inhibitory effect on osteogenesis-related protein expression in OS cells.Supplementary Table 4 presented the corresponding statistical data.

Real-time quantitative reverse transcription
The PCR results showed that the mRNA expression levels of BALP and CICP were inhibited in the Niraparib group, Anti-GD2 group, and Niraparib + Anti-GD2 group compared to the Control group.However, combining Niraparib with Anti-GD2 antibody had the most significant inhibitory effect on the mRNA expression levels of BALP and CICP in OS cells.On day 14, the absorbance of mRNA expression for BALP and CICP was measured; it was 0.173 ± 0.065 and 0.170 ± 0.078 respectively for the Niraparib + AntiGD2 group compared to Control group (both p < 0.01), indicating a statistically significant difference between them.These results demonstrate that combining Niraparib with Anti-GD2 antibody has a significant inhibitory effect on osteogenesis-related RNA expression in OS cells (Fig. 5).Supplementary Table 5 presented the corresponding statistical data.their inhibitory effects on BALP and CICP expression levels.In addition, we verified that GD2 expression was inhibited in the group with anti-GD2 antibody in both cell lines (Fig. 6).

The establishment of the subcutaneous osteosarcoma model
The results of animal experiments showed that tumor growth was inhibited in the Niraparib group, Anti-GD2 group, and Niraparib + Anti-GD2 group compared to the Control group.The Niraparib + Anti-GD2 group exhibited the most pronounced inhibitory effect on tumor growth.On the fifth day, the tumor volume of the Control group was 2433 ± 391, while that of the Niraparib + Anti-GD2 group was 1137 ± 148 (p < 0.01), indicating a statistically significant difference.
Combining Niraparib with an Anti-GD2 antibody demonstrated the most significant inhibition of subcutaneous tumor growth (Fig. 7).

Discussion
The combined use of PARP inhibitors and GD antibodies has already been observed in the field of tumor treatment, and we have summarized this information in Table 3.
The results of scratch and Transwell experiments confirmed that the Anti-GD2 antibody has an effect on the invasion ability of OS cells.In the scratch test, cells in the Anti-GD2 group crawled slower than those in the Control group.In the Transwell experiment, the Anti-GD2 group showed less cell invasion compared to the Control group.Previous studies have also demonstrated a correlation between Anti-GD2 antibody and aggressive tumor cell activity [13][14][15][16][17], which we confirmed in OS cells.According to preclinical studies, PARP inhibitors have potential therapeutic value for osteosarcoma (OS) [17][18][19][20][21].By screening for genetic determinants of drug activity, PARP inhibitors are shown to be potential targets for OS [21][22][23][24][25].The researchers also demonstrate that PARP inhibitors can enhance the activity of cytotoxic drugs [25][26][27][28][29].In our study, it was confirmed by a CCK8 experiment that Niraparib had a certain effect on cell viability.Furthermore, the cell viability of the Niraparib + Anti-GD2 group was significantly reduced, indicating that Niraparib enhanced the effect of Anti-GD2 antibody.
The preclinical studies indicate that OS cells are sensitive to PARP inhibitors in vitro [29][30][31][32][33][34][35], but the efficacy of PARP inhibitors as monotherapy in OS is limited, highlighting the need for combination therapy.PARP inhibitors enhance DNA damage-mediated cytotoxicity caused by topoisomerase I poisons, DNA methylators, or radiation, which is relevant to the role of PARP in repairing DNA damage caused by these cytotoxic therapies [36][37][38][39][40].In our study, osteogenesis was observed by detecting the expression of BALP and CICP.The results also confirmed that the effect of Niraparib monotherapy was limited.However, Niraparib could enhance the effect of Anti-GD2 antibody, and significantly reduce the expression of proteins associated with osteogenesis.
Our study emphasizes the necessity of combining PARP inhibitors with other drugs when monotherapy is ineffective in treating tumors.The study on the combination of PARP inhibitor and Anti-GD2 antibody in OS treatment demonstrated that Niraparib combined with Anti-GD2 antibody has a certain efficacy in treating OS.
It is crucial for us to emphasize this point here.We agree that subsequent treatments should be complemented by preclinical and even clinical studies.Although there are numerous potential targets for OS, our research on them remains limited.Therefore, we assert that it is premature to transition from cell experiments to clinical trials based solely on this study.The design of this study still has significant flaws, and we will gradually refine our conclusions in future research.

Conclusion
By studying the inhibitory effect of Niraparib alone or in combination with an Anti-GD2 antibody on OS, we found that the combined treatment of Niraparib and Anti-GD2 antibody had a more prominent inhibitory effect on OS compared to Niraparib alone.This provides a novel approach for treating OS.Subsequent clinical trials will be conducted to evaluate the inhibitory effect of combining Niraparib with an Anti-GD2 antibody on OS, with the expectation of making a significant contribution to the clinical treatment of this disease.

Fig. 2 Fig. 3
Fig. 2 Two cell lines' Transwell experiment results of Control group, Niraparib group, Anti-GD2 group and Niraparib + Anti-GD2 group were recorded.a MG63's transwell experiment results of Control group.b MG63's transwell experiment results of Niraparib group.c MG63's transwell experiment results of GD2 group.d MG63's transwell experiment results of Niraparib + GD2 group.e U2OS's transwell experiment results of Control group.f U2OS's transwell experiment results of Niraparib group.g U2OS's transwell experiment results of GD2 group.h U2OS's transwell experiment results of Niraparib + GD2 group.The experiment was repeated three times, with 8 samples each time

Fig. 4 Fig. 5
Fig. 4 Two cell lines' western blotting results of Control group, Niraparib group, Anti-GD2 group and Niraparib + Anti-GD2 group.The expression levels of BALP and CICP were detected with GAPDH as internal reference.A MG63's western blotting results of Control group, Niraparib group, Anti-GD2 group and Niraparib + Anti-GD2 group.B U2OS's western blotting results of Control group, Niraparib group, Anti-GD2 group and Niraparib + Anti-GD2 group.C MG63's target protein expression of semi-quantitative analysis.(D) U2OS's target protein expression of semi-quantitative analysis.(gray square) Relative value of Niraparib and Control group.(red square) Anti-GD2 and Control group.(green square) Niraparib + Anti-GD2 and Control group.The experiment was repeated three times, with 24 samples each time

Fig. 7 A
Fig. 7 A Images of a subcutaneous osteosarcoma model.B The results of tumors volume in each group.(a) Images of a subcutaneous osteosarcoma model in Control group.(b) Images of a subcutaneous osteosarcoma model in Niraparib group.(c) Images of a subcutaneous osteosarcoma model Anti-GD2 group.(d) Images of a subcutaneous osteosarcoma model in Niraparib + Anti-GD2 group.The experiment was repeated three times, with 12 samples each time

Table 2
The data of OD value in each group

Table 3
Combined use of PARP inhibitors and GD antibodies on tumors Author