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. 2024 Oct 13;13(5):tfae172. doi: 10.1093/toxres/tfae172

Reproductive safety of STING agonists MSA-2 and manganese-MSA-2

Ya Cai 1,#, Tian He 2,#, Tao Yang 3, Yating Li 4, Lirong Yi 5, Wenqing Li 6,✉, Peng Zhou 7
PMCID: PMC11471313  PMID: 39403122

Abstract

Background

MSA-2, as an oral molecule for activating STING signaling pathway to cure the tumor entering clinical trials. The toxicity of MSA-2 has aroused wide concern, especially the reproductive toxicity can not be ignored.

Objectives

We synthesized the STING agonist (MSA-2) and its derivative manganese-MSA-2 (MSA-2-Mn) and investigated the reproductive toxicity.

Methods

We evaluated the reproductive effects of MSA-2 and MSA-2-Mn in female mice under the administration alone and on the reproductive system of male mice in the presence or absence of combined radiation.

Results

Results suggested that MSA-2 and MSA-2-Mn have negligible reproductive toxicity in healthy adults. Conclusions: This provides new ideas to enhance the efficacy of immunotherapy, as well as favorable evidence for future systemic dosing in patients of reproductive age and clinical trials of immunotherapy.

Keywords: STING agonist, MSA-2, MSA-2-Mn, radiation, reproductive toxicitys

Introduction

Intrinsic immune system regulated by pharmaceutical stimulation of interferon gene-stimulating factor (STING) has been a promising method for tumor therapy.1–6 Conventional cyclic dinucleotides (CDNs) as natural STING agonists possess strong anticancer effects when administered intratumorally alone or combined with PD-1 or PD-L1 inhibitors.7–9 Recently, MSA-2, an orally available non-nucleotide of human STING agonist was discovered for tumor immunotherapy due to its small size, and cell permeability, which reveals extensive application prospects in tumor immunotherapy.10–14 In addition, manganese ions (Mn2+) are effective activators of the cGAS-STING pathway, which promotes CD8+ T-cell activation and enhances tumor therapeutic effects.15–18

Immunotherapy has made tremendous advances that have transformed traditional clinical treatments. However, it is known very little about the reproductive toxic effects of novel immunotherapies.19 Research has reported that immune checkpoint inhibitors, including nivolumab and Pembrolizumab (PD-1 inhibitors), atezolizumab (PD-L1 inhibitor), and ipilimumab (Cytotoxic T Lymphocyte-Associated protein-4 inhibitor),20 increased ovarian immune cell infiltration and tumor necrosis factor-α (TNF-α) expression, leading to ovulatory dysfunction and infertility, which indicated the potential reproductive toxicity of immunotherapies in female patients.21 Therefore, it is necessary to investigating the immune effect and immune toxicity of STING agonists MSA-2 and MSA-2-Mn.

Here, we synthesized a STING agonist MSA-2 and its derivative manganese-MSA-2 (MSA-2-Mn). We investigated the reproductive toxicity of MSA-2 and MSA-2-Mn in female and male mice. Furthermore, we investigate the combination of radiation (IR) and MSA-2 or MSA-2-Mn in male mice.22,23 We found that did not impact the oogenesis and spermatogenesis in female and male mice after MSA-2 and MSA-2-Mn, radiation combined MSA-2 and MSA-2-Mn treatment. The results demonstrated that MSA-2 and MSA-2-Mn were safe for use in tumor therapy and were of great benefit in advancing the clinical application of tumor immunotherapy.

Materials and methods

Mice

Male and Female C57BL/6J mice, approximately 8 weeks old, were obtained from the animal center of Nantong University, Nantong, China. The mice were housed under SPF standard laboratory conditions with a 12-h light–dark cycle and free access to food and water. All experimental procedures complied with the guidance of the Institutional Animal Care and Use Committee of the Nantong Medical School, Nantong University, China.

Synthesis of the MSA-2-Mn

An aqueous solution of 0.2 mmol NaOH was added to 0.2 mmol of 4-(5,6-dimethoxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid dissolved in the minimum of distilled water. The mixture was kept in the dark, and a solution of 0.1 mmol MnCl2 was added which yielded a precipitate. The heterogeneous mixture was stirred for 24-48 h and the products were isolated by filtration and stored in a desiccator.

MSA-2 and MSA-2-Mn administration in female mice

Twenty-five C57BL6/J female mice (8 weeks) were divided randomly into five groups: control, 20 mg/kg MSA-2, 50 mg/kg MSA-2, 20 mg/kg MSA-2-Mn, and 50 mg/kg MSA-2-Mn groups, with 5 animals in each group. Periodically monitored the body weight of the mice and carefully observed their daily food consumption and activity status throughout the experimental procedure. The mice were given subcutaneous injections of 100 μL volume of the corresponding concentration of the drug on days 1, 4, 7, 10, and 13, while the control group was administrated with a subcutaneous injection of 100 μL volume of the solvent (10% DMSO +40% PEG300 +50% Saline) (Supplementary Table S1 or Fig. 2A). Mice were sacrificed on day 21 after the first dose. The ovaries were collected for the following investigation.

Fig. 2.

Fig. 2

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A) Illustration for investigating the toxic effects of MSA-2 and MSA-2-Mn on female mice. Subcutaneous injection with 20 mg/kg and 50 mg/kg MSA-2 or MSA-2-Mn, equal volumes of solvent as control, respectively. Sacrificed on day 21. B) Body weight of control, 20 mg/kg MSA-2, 50 mg/kg MSA-2, 20 mg/kg MSA-2-Mn, and 50 mg/kg MSA-2-Mn. Data presented mean ± S.D. (n = 5). C) Relative organ weight (ovary/ body weight (mg/g)) of control, 20 mg/kg MSA-2, 50 mg/kg MSA-2, 20 mg/kg MSA-2-Mn, and 50 mg/kg MSA-2-Mn. Data presented mean ± S.D. (n = 5). D) The morphology of the ovary staining by Hematoxylin and eosin stain (H&E stain) (n = 5). E) Primordial follicles, primary follicles, secondary follicles, antral follicles, and corpora lutea were analyzed in Fig. 2D. (n = 5). F) TUNEL assay of the ovary in each group (n = 5). G) Concentrations of FSH, LH, and E in the plasma of female mice. Subcutaneous injection with 20 mg/kg and 50 mg/kg MSA-2 or MSA-2-Mn, equal volumes of solvent as control, respectively. Sacrificed on day 21. Data presented mean ± S.D. (n = 5).

Determination of relative organ weight

The mice were weighted. Then, the mice were sacrificed, the abdominal cavity was quickly dissected, and both ovaries were collected. The relative organ weight of the mouse ovaries was calculated according to the following formula: organ coefficient (ovary/ body weight (mg/g)) = weight of ovary (mg)/body weight (g).

Histological analysis of the ovaries

Mouse ovaries were fixed with MDF tissue fixative, paraffin-embedded, sectioned, and stained with a hematoxylin–eosin (H&E) staining kit (E607318, Sangon Biotech, China). Images were acquired under a light microscope (Leica DM2000, Germany).

Number of follicles

Twenty consecutive 5 μm sections of 1 paraffin-embedded ovarian tissue were taken from each animal, 1 section was taken every 5 sections for HE staining, and 5 sections were taken from each ovarian tissue to count the number of follicles and corpus luteum at all levels, and oocytes in the follicles in each section were counted as if there were nuclei in the follicles, to avoid double counting. The classification criteria of follicles at all levels were referred to in the literature.24,25

TUNEL assay

Mouse ovaries were fixed with MDF tissue fixative, paraffin-embedded, and sectioned. (1) Paraffin sections were dewaxed to water. (2) Wipe off excess water from the slices, and add DNase-free proteinase K 20 μg/mL dropwise (diluted 1,000-fold with 10 mM Tris–HCl pH = 7.4–7.8). (3) Wash the slices with PBS for 5 min each time, and wash them 3 times. (4) Prepare TUNEL assay solution: 5 μL of TdT enzyme was added to 45 μL of fluorescent labeling solution, and 50 μL of TUNEL assay solution was added dropwise to each sample. (5) Incubate at 37 °C for 1 h, avoiding light. (6) Wash with PBS for 5 min each time, 3 times in total. (7) Nuclei were stained using DAPI (Abcam, ab104139) to cover the slides. (8) A fluorescence microscope (ZEISS, Oberkochen, Germany) was used to acquire images.

Measurement of plasma hormone levels

On the 21 day after the initial administration, Mice were anesthetized using 0.2 mL of Avertin (from Nanjing Aibei, China) per 10 grams of their body weight, administered intraperitoneally. The effectiveness of the anesthesia was verified by the absence of responses to toe pinches, loss of the righting reflex, and a decrease in breathing rate. When the mice showed no reaction to a toe pinch for about 5 min, we begin to collect blood through orbital sinus puncture. Followed by cervical dislocation for euthanasia.

The blood was collected into a 2 mL centrifuge tube and centrifuged at 4,500 r/min for 10 min at 4 °C. The centrifuge tube was removed, and the upper layer of serum was pipetted into a new centrifuge tube, labeled, and stored in a refrigerator at −20 °C. The blood was stored at −20 °C. The levels of Estrogen (E), Follicle-Stimulating Hormone (FSH), and Luteinizing Hormone (LH) in mouse serum were determined by double-antibody sandwich ELISA, and the experimental procedure of ELISA was carried out according to the instructions of the kit (the mouse E ELISA Kit (Cat: MM-0546M1, Meimian, China), the mouse FSH ELISA Kit (Cat: MM-45654M1, Meimian, China), the mouse LH ELISA Kit (Cat: MM-0582M1, Meimian, China)).

Fertility assay

Fifteen C57BL6/J female mice (8 weeks) were randomly divided into control, 50 mg/kg MSA-2, and 50 mg/kg MSA-2-Mn groups. The mice were subcutaneously injected with the corresponding concentration of the drug on days 1, 4, 7, 10, and 13, while the control group (10% DMSO +40% PEG300 +50% Saline). On the 21 days after the initial administration, ordered a total of healthy eight 8-week male mice and conducted fertility experiments according to the ratio of two females and one male (Supplementary Table S2 or Fig. 3A). The female mice were observed for a few consecutive days, confirm successful mating in female mice by checking for the presence of a vaginal plug outside their external genitalia. The pregnancy rate was counted, and the male mice were separated immediately. Females were allowed to deliver normally, and the litters were processed. Pups were counted. On postnatal day 21, both the adult females and their pups were sacrificed using CO2 treatment.

Fig. 3.

Fig. 3

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Fertility of the mice treated with MSA-2 and MSA-2-Mn. A) Illustration for investigating the toxic effects of MSA-2 and MSA-2-Mn on female mice. Subcutaneous injection with 50 mg/kg MSA-2 or MSA-2-Mn, equal volumes of solvent as control, respectively. Mated with male mice on day 21. B) Fertility index and C) the average number of pups/pregnant females on days 21 (n = 5). Data showed mean ± S.D. Fertility index (%) = (no. of pregnant females/no. of females with successful copulation) × 100. The average number of pups per pregnant female = (no. of pups/ no. of pregnant females) × 100.

MSA-2 and MSA-2-Mn alone and in combination with IR administration in male mice

Fifteen C57BL6/J male mice (11 weeks) were randomly divided into three groups: control, MSA-2 and MSA-2-Mn groups, with 5 animals in each group. On days 1, 4, 7, 10, and 13, the experimental groups were injected with 100 μL of 50 mg/kg MSA-2 and MSA-2-Mn, and the control group was injected with 100 μL of a solvent (10% DMSO +40% PEG300 +50% Saline) subcutaneously. Mice were executed on day 14. Testis and epididymis were collected for the following investigation (Supplementary Table S3 or Fig. 4A).

Fig. 4.

Fig. 4

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Toxicity of MSA-2 and MSA-2-Mn on the reproductive system of male mice. A) Schematic diagram of the toxic effects of MSA-2 and MSA-2-Mn on male mice. Mice were divided into three groups of control, MSA-2 and MSA-2-Mn, and injected subcutaneously with 50 mg/kg of MSA-2 or MSA-2-Mn, and an equal volume of solvent as control, and injected once every 3 days for 5 consecutive injections, and then the mice were put to death on day 14. B) Body weight of control, MSA-2 and MSA-2-Mn. Data presented mean ± S.D. (n = 5). C) Schematic diagram of testicular morphology and size. D) Testicular index (testis/body weight (mg/g)). Data are mean ± S.D. (n = 5). E) Morphology of hematoxylin–eosin staining (H&E staining) of testis (n = 5). F) Morphology of hematoxylin–eosin staining (H&E staining) of epididymis (n = 5). G) Effects of MSA-2 and MSA-2-Mn on sperm quality in male mice. Computer-assisted sperm analyzer images (Above). Sperm concentration, viability and velocity in each group (n = 5) (Below).

Fifteen C57BL6/J male mice (11 weeks) were randomly divided into three groups: IR, IR-MSA-2 and IR-MSA-2-Mn groups, with 5 animals in each group. All mice were treated with 4 Gy X-ray irradiation 1 day before drug administration. The experimental groups were injected with 100 μL of MSA-2 and MSA-2-Mn at 50 mg/kg subcutaneously on days 1, 4, 7, 10, and 13, respectively, and the control group was injected with 100 μL of a solvent (10% DMSO +40% PEG300 +50% Saline) subcutaneously on days 1, 4, 7, 10, and 13, respectively (Supplementary Table S4 or Fig. 5A). Mice were executed on day 14. Testis and epididymis were collected for the following investigation. Periodically monitored the body weight of the mice and carefully observed their daily food consumption and activity status throughout the experimental procedure.

Fig. 5.

Fig. 5

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Toxicity of MSA-2 and MSA-2-manganese in combination with radiotherapy on the reproductive system of male mice. A) Schematic diagram of the toxic effects of MSA-2 and MSA-2-Mn in combination with radiotherapy on male mice. Mice were divided into three groups of IR, IR + MSA-2 and IR + MSA-2-Mn, and were first irradiated with X-rays at a dose of 4 Gy. Then, 50 mg/kg MSA-2 or MSA-2-Mn was injected subcutaneously, and an equal volume of solvent as control, and the mice were injected once every 3 days for 5 consecutive injections, and then executed after 1 day. B) Body weight of IR, IR-MSA-2 and IR-MSA-2-Mn. Data presented mean ± S.D. (n = 5). C) Schematic diagram of testicular morphology and size. D) Testicular index (testis/body weight (mg/g)). Data are mean ± S.D. (n = 5). E) Morphology of hematoxylin–eosin staining (H&E staining) of testis (n = 5). F) Morphology of hematoxylin–eosin staining (H&E staining) of epididymis (n = 5). G) Effects of MSA-2 and MSA-2-Mn and their combination with radiotherapy on sperm quality in male mice. Computer-assisted sperm analyzer images (Above). Sperm concentration, viability, and velocity in each group (n = 5) (Below).

Histological analysis of the testis and epididymis

Mouse testis and epididymis were fixed with MDF tissue fixative, paraffin-embedded, sectioned, and stained with a hematoxylin–eosin (H&E) staining kit (E607318, Sangon Biotech, China). Images were acquired under a light microscope (Leica DM2000, Germany) or Olympus FV3000 (Japan).

Determination of relative organ weight (testis/body weight (mg/g))

The mice were weighted. Then, the mice were sacrificed, the abdominal cavity was quickly dissected, and both testis were collected. The relative organ weight of the mouse testis was calculated according to the following formula: The relative organ weight (testis/body weight (mg/g)) = weight of testis (mg)/body weight (g).

Motility parameters of sperm by CASA.

1 mL Tyrode solution (PB180338, Procell) was preheated in EP tube, metal bath (Thermo Science, U.S.A) heated to 37 °C, Sacrifice mice, the epididymis was cut in EP tube, cut and incubated at constant temperature for more than 10 min. The data was detected by IVOS CASA system (Hamilton Thorne, U.S.A). During this period, the real-time state of the sperm is captured.

Elisa for plasma IL-6,TNF-α and IFN-γ.

Reagent source: IL-6 (RayBio,ELM-IL6),TNF-α (R&D SYSTEMS, MIF00-1), IFN-γ (R&D SYSTEMS, MTA00B) The mice were anesthetized with afudine anesthetic, underwent orbital blood collection and waited for natural blood clotting for about 30 min, and centrifuged at 10,000 rpm for 20 min. Remove the supernatant and replace it with negative 80 °C. Dilute standard. Samples were taken and thawed, added to a 96-well plate and mixed well. Incubation was performed according to the instructions. The wash solution was diluted with distilled water, washed with the diluted solution, and dried. Add the corresponding enzyme reagent. The temperature incubation and washing were repeated. Color-gen was added and incubated according to the instructions. The reaction was stopped by adding the stop solution. The OD value at 450 nm was measured by a microplate reader, and the standard curve was drawn according to the measured value to calculate the specific value.

Blood testing

Mice were anesthetized with avudine anesthesia, orbital blood sampling was performed, and blood was put in an anticoagulant tube, and sent to the testing institution for routine blood test within 1 h.

Statistical analysis

All data were statistically analyzed and plotted using GraphPad Prism 9.5 software, and a two-sided t-test or One-way ANOVA was used for statistical analysis between samples, displaying the data as the standard deviation of the mean, P < 0.05 indicated that the differences were statistically significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Results and discussion

Synthesized STING agonist MSA-2 and its derivative manganese-MSA-2 (MSA-2-Mn).

An orally available non-nucleotide human STING agonist MSA-2 was discovered for tumor immunotherapy with its smaller size and higher cell permeability than cyclic dinucleotides (CDNs).13 Also, recent studies have reported that Mn2+ sensitized the cGAS-STING pathway, and amplified STING activation, which might show that Mn2+ would enhance the STING activation of MSA-2.18 Therefore, we synthesized manganese-modified MSA-2 (MSA-2-Mn) from MSA-2. The synthesis steps were as in Fig. 1A, and the 1H NMR and 13C NMR Spectrum data showed the characterizations of MSA-2-Mn (supplementary Information), and the MS supplementary Fig. S1). Then, we investigated the immnue toxicity in mice as the research program including the structures of MSA-2 and MSA-2-Mn (Left), reproductive toxicity of MSA-2 and MSA-2-Mn, radiation combined MSA-2 and MSA-2-Mn treatment (Right) (Fig. 1B). In addition, we have conducted experiments on the STING immune activation effect, the results showed that MSA-2 and MSA-2-Mn can induce the immune activation, especially with MSA-2 (supplementary Fig. S2).

Fig. 1.

Fig. 1

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A) Synthesis of the MSA-2-Mn form MSA-2. B) Research program including the structures of MSA-2 and MSA-2-Mn (Left), reproductive toxicity research of MSA-2 and MSA-2-Mn, and radiation combined MSA-2 and MSA-2-Mn (Right).

The MSA-2 and MSA-2-Mn did not affect the oogenesis in female mice

In order to study the toxic effects of MSA-2 and MSA-2-Mn on female reproductive systems, adult C57BL6/J female mice were subcutaneously (s.c.) injected with the doses of 20 mg/kg and 50 mg/kg with MSA-2 or MSA-2-Mn every 3 days for five doses, equal volumes of solvent as control, respectively. Then, the ovaries were collected 21 days after the initial administration, which represented one follicular cycle (the period for an immature primordial follicle to develop into a mature ovulating oocyte)26 (Fig. 2A). During the experimental period, the mice were observed and weighed. We found that the body weight was decreased a little with 50 mg/kg MSA-2 treatment. There were no significant changes in each group (Fig. 2B). The relative organ weight (ovary/body weight (mg/g)) of each group showed no significant differences in female mice compared with those of the control group (Fig. 2C).

The ovaries were morphologically smaller in both high-dose immunotherapy groups compared to the control group. The number of primordial follicles, primary follicles, and corpora lutea decreased in the immunotherapy groups, while the number of secondary follicles and antral follicles increased (Fig. 2D and E). Ovaries from both MSA-2 and MSA-2-Mn treatment groups had no significant cell apoptosis compared with the control group (Fig. 2F). The immunotherapy group exhibited a decrease in the number of primordial follicles, primary follicles, and corpora lutea; however, there was no statistically significant difference when compared to the control group and the experimental group ovaries showed no significant apoptosis. These results indicated that MSA-2 and MSA-2-Mn did not affect the oogenesis of female mice.

FSH, LH, and E are all known to be important hormones that influence follicular development and ovulation. The main function of follicle-stimulating hormone (FSH) is to stimulate follicle development and maturation, especially pre-sinus follicles and sinus follicles, by directly acting on receptors on granulosa cells.27–30 Luteinizing hormone (LH) stimulates the synthesis of androgens and provides a substrate for estrogen, which is capable of stimulating the development and maturation of oocytes before ovulation and facilitating follicular discharge.31,32 Furthermore, Estrogen (E), promotes follicular development and, in concert with FSH, promotes the synthesis of LH receptors by follicular endothelial cells and granulosa cells to support LH regulation of follicular secretion.33,34 Therefore, we examined the serum levels of FSH, LH, and E in mice, 21 days after the initial administration. We found that the serum concentrations of FSH, LH, and E increased proportionally in all treatment groups compared to the control group. Among them, the increase in serum luteinizing hormone (LH) was more significant in the 50 mg/kg MSA-2 and 20 mg/kg MSA-2-MN treatment groups. (Fig. 2G). The follicle-stimulating hormone (FSH) and estrogen (E) showed no significant differences between the MSA-2 and MSA-2-Mn treatment groups compared with the control group (Fig. 2G).

Finally, the fertility and the average number of pups of mice were evaluated on day 21 with MSA-2 and MSA-2-Mn treatment (Fig. 3A). In Fig. 3B, the fertility index for the MSA-2-Mn group is shown to be 80%. However, statistical analysis indicated that there were no significant differences in the fertility indices among the three groups.

In addition, no significant differences in the average number of pups among groups after MSA-2 and MSA-2-Mn treatment (Fig. 3C). The results indicated that bearable reproductive toxicity of female mice was caused by the STING agonist MSA-2 and its derivative MSA-2-Mn.

No reproductive toxicity of MSA-2 and MSA-2-Mn in male mice

To investigate the toxic effects of MSA-2 and MSA-2-Mn on the male reproductive system, C57BL6/J adult male mice were injected subcutaneously with MSA-2 and MSA-2-Mn at a dose of 50 mg/kg respectively, every 3 days for five doses. An equal volume of solvent as control, and testes and epididymis were collected on day 14 (Fig. 4A). The experimental results indicate that there was no statistically significant change in the body weight of the mice. (Fig. 4B). We found that the morphology and weight of testes were the same in all groups, and the organ coefficients (testes/body weight (mg/g)) were not significantly different from those of the control group (Fig. 4C and D). The testicular seminiferous tubules and epididymal output tubules of mice in all groups were neatly arranged and uniformly distributed, and the spermatogenic cells were abundant in number and normal in morphology (Fig. 4E and F). These results indicated that MSA-2 and MSA-2-Mn were not significantly toxic to the reproductive structures of male mice. On day 14, we also assessed sperm quality in mice treated with MSA-2 and MSA-2-Mn. We found no significant differences in sperm concentration, viability and velocity in the other groups compared to the blank control group (Fig. 4H). Under the circumstances of the study, the findings revealed that both MSA-2 and MSA-2-Mn exerted no significant influence on sperm quality in male mice.

Combinding radiotherapy with MSA-2 or MSA-2-Mn generated no additional reproductive toxicity in male mice

In addition, the combination of radiotherapy and immunotherapy not only produces a synergistic effect in tumors, but also produces the toxic side effects. Therefore, we further investigated the effects of radiotherapy in combination with MSA-2 and MSA-2-Mn on the male reproductive system.22,23 We first treated all C57BL6/J adult male mice with radiotherapy with 4 Gy X-ray irradiation and exposed duration of 2.7 min, and similarly injected MSA-2 and MSA-2-Mn subcutaneously at a dose of 50 mg/kg, respectively, every 3 days for five doses. Equal volumes of solvents were used as a control, and testes and epididymis were collected on the 14th day (Fig. 5A). The data revealed no statistically significant alteration in the body weight of the mice (Fig. 5B). We found that the testicular morphology and relative testis weight (testis/body weight (mg/g)) became smaller in all groups of mice treated with radiotherapy, whereas there was no significant difference between the combined treatment group and the radiotherapy alone group (Fig. 5C and D). In addition, interstitial cell infiltration was seen in the testicular convoluted tubules, with irregular morphology of the supporting cells, and vacuolar degeneration and contraction were seen in the radiotherapy alone group, and thinning of the epididymal output tubule wall and enlargement of the lumen were seen. And equally, there was no significant difference between the combined treatment group and the radiotherapy alone group. (Fig. 5E and F). These results indicate that the combination treatment of STING agonists MSA-2 and MSA-2-Mn with radiotherapy does not enhance the damage of radiotherapy to the reproductive system in male mice. As well, we also assessed sperm quality in mice treated with MSA-2 and MSA-2-Mn combined with radiotherapy on day 14. We found no significant differences in sperm concentration, viability, and velocity in the other groups compared to the blank control group (Fig. 5H). During the course of the experiment, we conducted observations on the mice, which included routine blood tests. The granulocytes, red blood cells (RBCs), and platelets (PLT) were analyzed, and statistical analysis revealed no significant differences among the groups. (Supplementary Fig. S3). The results indicated that MSA-2 and MSA-2-Mn in combination with radiotherapy had no significant effect on sperm quality in male mice.

Conclusion

In summary, we synthesized a STING agonist (MSA-2) and its derivative manganese-MSA-2 (MSA-2-Mn). In addition, we conducted a comprehensive study in healthy adult mice in which the STING agonist MSA-2 and its metal derivative MSA-2-Mn caused no significant reproductive damage in either female or male mice, and the combination of radiotherapy did not produce additional reproductive toxicity in male mice. As demonstrated in our study, the reproductive toxicity associated with this immunotherapy is acceptable, with fertility preserved in reproductive-age cancer survivors treated with this therapy.

Supplementary Material

Supporting_information-2024-9-9_tfae172
supporting_information-2024-9-9_tfae172.docx (243.1KB, docx)

Acknowledgments

The authors thank all study participants for their support of the study. We also thank Jiangsu Specially-Appointed Professor (06200054, W. L.), the Science and Technology Program of Nantong City (JC2021057, L. Y.), and the start-up fund from Nantong University for funding this study.

Contributor Information

Ya Cai, Institute of Reproductive Medicine, School of Medicine, Nantong University, No. 16 Wenfeng Road, Chongchuan District, Nantong 226000, China.

Tian He, Institute of Reproductive Medicine, School of Medicine, Nantong University, No. 16 Wenfeng Road, Chongchuan District, Nantong 226000, China.

Tao Yang, Department of Radiotherapy, Affiliated Hospital of Nantong University, No. 20 Xisi Road, Chongchuan District, Nantong, Jiangsu 226000, China.

Yating Li, Institute of Reproductive Medicine, School of Medicine, Nantong University, No. 16 Wenfeng Road, Chongchuan District, Nantong 226000, China.

Lirong Yi, Institute of Reproductive Medicine, School of Medicine, Nantong University, No. 16 Wenfeng Road, Chongchuan District, Nantong 226000, China.

Wenqing Li, Institute of Reproductive Medicine, School of Medicine, Nantong University, No. 16 Wenfeng Road, Chongchuan District, Nantong 226000, China.

Peng Zhou, Institute of Reproductive Medicine, School of Medicine, Nantong University, No. 16 Wenfeng Road, Chongchuan District, Nantong 226000, China.

Author contributions

Ya Cai: Investigation, Writing—original draft, Writing—review & editing, Submission, Visualization; Tian He: Investigation, Writing—original draft, Writing—review & editing, Submission, Visualization; Tao Yang: Resources (provision of experimental environment), Infrastructure; Peng Zhou: Material preparation (synthesis of experimental materials); Wenqing Li: Funding acquisition, Writing—review & editing (for review process), Methodology, Data Curation; Lirong Yi: Conceptualization, Supervision, Project Administration, Methodology; Yating Li: Investigation (assisted with experiments).

Supplementary material

Characterization data of 1H NMR spectrum, 13C NMR spectrum, IR spectrum, and HRMS for MSA-2-Mn. MS results of MSA-2-Mn. Cytokine levels in mouse serum measured by ELISA. Blood test results in mice. Summary table (table summarizing the experimental treatments for each group of mice, consistent with the information depicted in the main text figures).

Funding

This work was supported by Jiangsu Specially-Appointed Professor (06200054, W. L.), the Science and Technology Program of Nantong City (JC2021057, L. Y.), and the start-up fund from Nantong University.

Conflict of interest statement. There were no competing interests to declare.

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