Whole Cancer Cell Vaccine Protects Ovarian Functions in Tumor-bearing Mice through Downregulating CXCL10

doi:10.21203/rs.3.rs-3838016/v1

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Whole Cancer Cell Vaccine Protects Ovarian Functions in Tumor-bearing Mice through Downregulating CXCL10

https://doi.org/10.21203/rs.3.rs-3838016/v1

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Young female patients with cancer are likely to become sub-fertile or infertile even if they ultimately overcome cancer through various therapies. Cancer immunotherapy has recently emerged as a promising novel therapy against cancers with high malignancy and lethality, but it is unclear whether cancer immunotherapy affects female fertility. This study employed MCA205 cell-allotransplanted B6 mice as a model to investigate whether two popular immunotherapies—PD-1 monoclonal antibody (PD-1) therapy and whole cancer cell vaccine (WCV) therapy—affect ovarian function. MCA205 allotransplanted (M) mice exhibited decreased follicle numbers at each stage, decreased proliferation, increased apoptosis, and a decreased oocyte maturation rate. WCV treatment significantly reversed these abnormalities, whereas PD-1 did not. RNA sequencing of the ovaries revealed that multiple differentially expressed genes (DEGs) were involved in inflammation pathways. Furthermore, cytokine microarray characterized CXCL10 with both biggest increment in M group and best rescue in WCV group. Next, CXCL10 antibody Immunoprecipitation in ovarian lysate and liquid chromatography-mass spectrometry (LC-MS) baited the only receptor IL18R1. Furthermore, we found that CXCL10 impaired ovarian function through three pathways: inducing ovarian fibrosis through CXCL10→IL18R1→p-JNK→COL1A1, promoting primordial follicle overactivation through CXCL10→IL18R1→p-AKT, and increasing ovarian inflammation through CXCL10→IL18R1→p-P65. Finally, we rescued the decreased ovarian function in the M group by blocking the CXCL10→IL18R1 pathway with CXCL10 antibody or a CXCL10–IL18R1 interface peptide, CIBB. This study provides mechanical evidence and translational strategies for WCVs to achieve the dual functions of suppressing tumor progression while protecting ovarian function.

Health sciences/Medical research/Experimental models of disease

Biological sciences/Cancer/Cancer therapy/Tumour vaccines

Whole cell vaccine

ovarian functions

tumor-bearing mice

CXCL10

IL18R1

Cancer immunotherapy is recognized as a fourth cancer treatment, along with surgery, radiation, and chemotherapy. Compared with traditional cancer immunotherapy, which simply activates systemic immune responses, current cancer immunotherapy is based on the understanding of tumor-induced immune escape mechanisms. By reversing immune escape, this novel immunotherapy can partially restore normal anti-tumor immune responses, which include a series of connective processes such as the release of tumor antigen, tumor antigen presentation, the priming and activation of effector T cells, T cell migration to the tumor tissue, T-cell infiltration in the tumor tissue, T cell recognition of tumor cells, and the removal of tumor cells. Currently, several strategies are undergoing translational studies, and some are already applied in clinical settings, including monoclonal antibody-based immune checkpoint inhibitors, therapeutic antibodies, cancer vaccines, cell therapy, and small molecule inhibitors (1–5).

Current cancer immunotherapies have been employed as single agents or in combination to treat clinical tumors, and they appear to be more effective and less toxic. For example, the PD-1 inhibitors pembrolizumab and nivolumab are FDA (Food and Drug Administration)-approved for advanced melanoma (6), non-small cell lung cancer (7), Hodgkin's lymphoma (8), and head and neck squamous cell cancer (9). Nivolumab is also approved for kidney (7) and urothelial cancers (10). In addition, the PD-L1 inhibitors atezolizumab and durvalumab have also entered phase III clinical trials for non-small cell lung cancer (11), bladder cancer (12), and other tumor types (11).

The population and proportion of women of childbearing age with tumorigenesis is increasing because of various intrinsic and extrinsic detrimental factors. Cancer itself (13) and traditional tumor therapy (14) have been reported to substantially impair female fertility. In many cases, women of childbearing age must choose between eradicating a tumor and maintaining their fertility. Fortunately, multiple studies have shown that current cancer immunotherapy does not substantially impair female fertility. For example, women with metastatic melanoma (15), gestational trophoblastic tumors (16), hematological tumors, and other diverse solid tumors (17) have succeeded in becoming pregnant and delivering a newborn after treatment with immunotherapy. However, previous studies have only examined pregnancy and delivery without analyzing ovary performance and oocyte quality, which determine the ovarian lifespan and physical health supported by the ovary-secreting hormones.

This study tested two immunotherapies—PD1 monoclonal antibody therapy and whole cancer cell vaccine (WCV; cancer cells treated with Mitoxantrone (MTA) and then injected into tumor-bearing mice)—on the ovarian performance, pregnancy, and fertility of tumor-bearing female mice. We found that WCV treatment, but not PD-1 antibody treatment significantly improved ovarian performance, including primordial follicle reserve and oocyte quality. We also explored the mechanism by which tumor invasion impairs ovarian function and how WCVs protect ovarian performance from tumor-induced damage.

Antibodies

Information on all commercial primary and secondary antibodies is included in Supplementary Table 1.1 and 1.2 respectively.

Rabbit anti-CXCL10 was produced against CLNPESKTIKNL by ZoonBio BioTech (Nanjing, China) and purified by affinity purification.

Mice Model

All experimental procedures involving animals were approved by the Institutional Animal Care and Use Committee (IACUC) of Nanjing Medical University (Approval No., IACUC-2205056) and Anhui Medical University (Approval No., LLSC-20232253). All mice were housed under standard specific pathogen-free (SPF) conditions of the animal core facility (ACF). To acquire the ovaries and other tissues, mice were anesthetized with CO₂ and then sacrificed by cervical dislocation. Three mouse models were created as follows.

1). WCV and PD-1 monoclonal antibody immunotherapy in B6 mice

Immunotherapy mouse modeling was established as published (18). Four groups were set up: the CTR group, the M (MCA205) group, the VM (WCV + MCA205) group, and the PM (anti-PD-1 antibody + MCA205) group.

For CTR group, female C57BL/6 mice aged 7–8 weeks was injected with 100 µL PBS s.c. (subcutaneous) once daily. For M group, female C57BL/6 mice aged 7–8 weeks received subcutaneous injections of 1 × 10⁶ MCA205 cells, a malignant cell line from mouse fibrosarcoma. Tumor diameters were measured with digital calipers at multiple time points (days 3, 5, 7, 9, and 11), and the tumor area (in mm²) was calculated with the following formula: Area = 3.14 × Width × Length/4. For VM group, on day − 12, tumor cells were pre-treated with 2 µM MTA (mitoxantrone) for 16 h (Whole cancer cell vaccine, WCV), washed with PBS, and injected s.c. (1 × 10⁶ WCV cells in 100 µL PBS) into naive C57BL/6 mice, then on day 0, tumor cells were injected as in M group. For PM group, on day 0, tumor cells were injected as in M group, then on day 5 (When tumor size reached 25–45 mm²), 8, 11 respectively (3 injections totally for one time of modeling), mice were injected i.v. (intravascularly) with 10 mg/Kg anti-PD-1 mAb (in 100 µL PBS, clone RMP1-14, BioXCell, New Hampshire, USA).

2). CXCL10 antibody injection in tumor-bearing mice

For this, three groups were set up: the CTR group, the M (MCA205) group, and the CM (anti-CXCL10 antibody + MCA205) group.

For CTR or M group, the treatment was as above. For CM group, on day 0, tumor cells were injected as above; then on day 5 (When tumor size reached 25–45 mm²), 8, 11 respectively (3 injections totally for one time of modeling), mice were injected i.v. (intravascularly) with 10 mg/Kg anti-CXCL10 antibody (in 100 µL PBS).

3). Competitive peptide injection in tumor-bearing mice

For this, three groups were set up: the CTR group, the M (MCA205) group, and the CI-M (CIBB + MCA205) group.

For CTR group, the treatment was as above. For M group, tumor cells were injected as above, plus 6 mg/Kg control CPP (cell-penetrating peptide) TAT (trans-acting activator of transcription of HIV, CYGRKKRRQRRR) injection daily (in 100 µL PBS). For CI-M group, on day 0, tumor cells were injected as above; then from the next day, mice were injected i.p. (intraperitoneally) with 6 mg/Kg CIBB peptide daily (in 100 µL PBS).

For CIBB design that inhibits the interaction between CXCL10 and IL18R1, the protein structure of CXCL10 interacting with IL18R1 was predicted by AlphaFold. The sequences in CXCL10 that bound to IL18R1 in the top two prediction models were the M1 sequence (CLNPESKTI) and the M2 sequence (IGKLEIIPASLSCPRVEIIATMK). The Flag sequence (DYKDDDDK) and TAT sequence were fused with the M1 or M2 sequence and synthesized by Shanghai Bootech Bioscience & Technology (Shanghai, China). The combination of M1 and M2 was named CIBB (CXCL10-IL18R1 Binding Blocker), which was dissolved in 10% DMSO (dimethyl sulfoxide, Sigma, Saint Louis, USA) to a stock concentration of 5 mg/mL. The stock peptide was diluted with PBS to a final concentration of 0.5 mg/mL, and the injection dosage was 6 mg/Kg.

RNA sequencing and analysis

RNA samples were obtained from the ovaries of the mice. The process involved isolating RNA, conducting high-throughput sequencing, and analyzing the data. This was performed by Seq Health Technology (Wuhan, China) following standard protocols. The library products were then sequenced on a DNBSEQ-T7 sequencer (MGI Tech, Shenzhen, China) with the PE150 model. The original sequence datasets were submitted to the NCBI Sequence Read Archive database and assigned an accession number. Specifically, for the RNA-seq in Fig. 3A, the accession number was GSE248829. Gene with an absolute value of the log2 (treated/control) ratio greater than or equal to 1.2 and a q-value of less than 0.001 was determined as a DEG (differentially expressed gene).

Multiplex immunoassay

Mutiplex immunoassay was operated by Shanghai Universal Biotech (Shanghai, China). Blood plasma was collected from the CTR group, the M group, the VM group, and the PM group (five repeats each group). 50 µL of the collected sample was utilized to measure cytokines using Luminex magnetic beads according to the Mouse LX-MultiDTM-31 protocol (Bio-RAD, Hercules, USA, Cat. No. 12009159). The information was gathered with a Bio-Plex 200 system (Bio-RAD) with high-throughput fluidics (HTF) and analyzed using Bio-Plex Manager software version 6.1 (Bio-RAD).

Tissue dissociation and single-cell suspension preparation

The ovarian tissues were preserved in the GEXSCOPE tissue preservation solution (Singleron Biotech, Nanjing, China) and shipped to the Singleron lab with ice pack. The specimens were washed 3 times with HBSS (Hanks Balanced Salt Solution, Gibco, Cat. No. 14025-076) and shred into 1–2 mm pieces. Then the tissue debris were submitted to the digestion with 2 mL GEXSCOPE tissue dissociation solution (Singleron) at 37°C for 15 min in 15 mL centrifuge tube with sustained agitation. Cells were filtered through 40-micron sterile strainers and centrifuged at 300 g for 5 min. Then the supernatant was removed, and the pellets were resuspended in 1 mL PBS. To remove the red blood cells, which were frequently a significant portion of the cells produced, 2 mL RBC lysis buffer (Roche, Indianapolis, USA, Cat. No. 11814389001) was added to the cell suspension according to the manufacturer’s protocol. Next, the cells were centrifuged at 500 × g for 5 min at 15–25°C and resuspend in PBS. The sample from the cell mixture was stained with trypan blue (Bio-RAD, Cat. No. 1450013), counted, and the cell density was adjusted to 1 × 10⁵ cells/mL. Once the cell viability exceeded 80%, subsequent sample processing could be performed.

Single-cell RNA sequencing and data analysis

Single-cell suspensions with 1 × 10⁵ cells/mL in PBS were prepared. Single-cell suspensions were then loaded onto microfluidic devices and scRNA-seq libraries were constructed according to Singleron GEXSCOPE protocol by GEXSCOPE Single-Cell RNA Library Kit (Singleron), which included cell disruption, mRNA trapping, cell labeling (with barcode) and mRNA labeling (with UMI), reverse transcription of mRNA into cDNA and amplification, cDNA fragmentation, and finally cDNA fragments cloning. Individual libraries were diluted to 4 nM and pooled for sequencing. Pools were sequenced on Novaseq 6000 (Illumina, San Diego, USA) with 150 bp paired end reads. Raw reads were processed with fastQC and fastp to remove low quality reads. Poly-A tails and adaptor sequences were removed by cutadapt. After quality control, reads were mapped to the reference genome GRCm38 (ensembl version 99 annotation) using STAR. Gene counts and UMI counts were acquired by featureCounts software. Expression matrix files for subsequent analyses were generated based on gene counts and UMI counts. We selected the genes expressed in more than 10% of the cells in either of the compared groups of cells and with an average log (Fold Change) value greater than 1 as DEGs. Adjusted p value was calculated by benjamini-hochberg correction and the value 0.05 was used as the criterion to evaluate the statistical significance.

Flow cytometric analysis

Six mouse ovaries were used for each sample. The freshly dissected ovaries were immediately placed into ice-cold PBS, then carefully dissected, and minced by scissors. Next, the ovaries were digested with 3 mL of digestion buffer containing RPMI-1640 medium, 4 mg Collagenase IV (Sigma, Saint Louis, USA, Cat No. AC5138), and 0.25 mg Deoxyribonuclease I (Sigma, Cat No. DN25) for 40 min on a shaker at 120 RPM and 37°C. Next, the digestion was stopped by 1 mL of PBS containing 20% FBS (fetal bovine serum).

The cell pellets were centrifuged 10 min at 600 g and 4°C, resuspended in FACS (Fluorescence-Activated Cell Sorting) buffer (PBS with 0.5% BSA (Bovine Serum Albumin) and 5 mM EDTA), and filtered through a 70 µm nylon mesh filter (Biosharp, Beijing, China, Cat No. BS-70-CS).

Cells were stained for live/dead viability using Zombie Violet™ Fixable Viability Kit (Biolegend, Beijing, China, Cat No. 423113) for 20 min, followed by incubation with anti-CD16/CD32 monoclonal antibody (Biolegend, Cat No. 156603) to block non-specific antibody staining. Cells were incubated with cell surface antigen-specific antibodies, such as CD45 antibody (Biolegend, Cat No. 103116), F4/80 antibody (Biolegend, Cat No. 123110), CD11b antibody (Biolegend, Cat No. 101206), and CD86 antibody (Biolegend, Cat No. 105014), on ice for 60 min. After washing, cells were retained approximately 100 µL residual volume, fixed with 100 µL IC fixation solution (Thermo Fisher, Waltham, USA, Cat No. 88-8824-00) for 60 min. Cells were washed twice with 2 mL permeabilization solution, then incubated with CD206 antibody (Biolegend, Cat No. 141712) for 60 min. Finally, cells were resuspended in 500 µL PBS and passed through a 70 µm nylon mesh filter for analysis on the Cytoflex LX cell analyzer (Beckman, Miami, USA) using CytExpert software.

After being gated, CD45+, F4/80 + and CD11b+, CD86+/CD206- cells were analyzed as M1 macrophage while CD86-/CD206 + cells were analyzed as M2 macrophage. The gating strategy is in supplementary Fig. 5.

Expression and purification of IL18R1 in SF9 cells

IL18R1 protein, fused with EGFP-Strep II, was cloned and expressed through a Bac-to-bac system (Themo Fisher). In short, the corresponding sequence (Supplementary Table 2) was cloned into pFastBacHTA (Supplementary table 4) and then transformed into DH10Bac (Vazyme, Nanjing, USA) competent E.coli. The bacmid was isolated from the E. coli using the QIAFILTER plasmid purification specimen (QIAGEN, Tegelen, Netherlands) and then transfected into Sf9 cells (Genetimes ExCell Technology, Shanghai, China, Cat. No. ATCC CRL-3357) with Cellfectin II Transfection reagent (Thermo Fisher) to produce first-round baculovirus. The fresh Sf9 cells were infected with the first-round baculovirus for 48 h for second- and third-round of virus amplification. Twenty µL of the third-round virus supernatant was used to infect 500 mL (SFM900-II medium, with 5% FBS, Thermo Fisher) Sf9 cells (1.5 x 10⁶/mL) for protein expression in an orbital shaker (Shanghai Zhichu Instrument) at 27°C and 200 RPM. The infected cells were resuspended in a lysis buffer (containing 50 mM Tris, 10% sucrose, 50 µM ATP, 1 mM phenylmethylsulfonyl fluoride (PMSF), 5 mM dithiothreitol (DTT), 1% NP40, 10 mM imidazole, 1 × protease inhibitor and phosphatase inhibitor, pH 7.0 by HCl). Cell lysate was further lysed with high-pressure cell disrupter (Union Biotech, Shanghai, China) and centrifuged; the lysate supernatant was incubated with 1 mL Ni-NTA Superflow resin (QIAGEN) for 1 h at 4°C. The resin was then transferred into a 5 mL chromatography column (Biocomma, Shenzhen, China) and washed with four column-volume of wash buffer (40 mM imidazole, with PMSF free). Finally, the protein was eluted with resuspension buffer (500 mM imidazole, without PMSF). The eluted protein was concentrated by a size-exclusion spin column and exchanged into BRB80 buffer (80 mM HEPES, 1 mM MgCl₂ and 1 mM EGTA, pH 6.8 by KOH) with 10% glycerol, 50 µM ATP and 5 mM DTT. The protein was aliquoted and kept at − 80 ˚C for future use. The conc. of IL18R1 was determined by comparing its intensity with 0.4 mg/mL BSA (Fig. 4C)

Expression and purification of CXCL10 in Ecoli, GST-pulldown, and mass spectrometry

The construct containing pGEX-6P1-CXCL10-StrepII was propagated in BL-21 E.coli (Vazyme, Cat. No. C504). 10 mL overnight culture was added into 1000 mL LB medium and the E.coli grew on an orbital shaker at 27°C and 200 RPM till the OD₆₀₀ reached 0.6. Then the E.coli culture was induced with 0.1 mM IPTG (Isopropyl-β-d-thiogalactoside, Yeasen, Shanghai, China, Cat. No. 10902ES10) at 16°C for 16 h. Next, the E.coli culture were collected by centrifugation for 10 min at 3500 RPM and 4°C, washed with ice-cold PBS at pH 7.3, and lysed in 25 mL lysis buffer (PBS, pH 7.3, 1 mM DTT (Dithiothreitol, Amresco, Framingham, USA, Cat. No. M109), 1 mM phenylmethylsulfonylfluoride (PMSF, Amresco, Cat. No. M145), 1:100 InStab™ protease inhibitor cocktail (Yeasen, Cat. No. 20124ES10), 1:100 InStab™ phosphatase inhibitor cocktail (Yeasen, Cat. No. 20109ES20), 1% Triton X-100). After standing on ice for 15 min, the E.coli were broken by a high-pressure crusher, and the supernatant was collected by centrifugation at 4°C. The fusion protein was captured with 600 µl glutathione agarose resin (BBI Life Science, Shanghai, China, Cat. No. C600031-0010) at 4°C for 30 min and then collected by low-speed centrifugation. To purify the fusion protein, the GST beads were washed with 20 mL washing buffer three times. The conc. of CXCL10 was determined by comparing its intensity with 0.4 mg/mL BSA (Fig. 3H).

To identify CXCL10-interacting proteins in ovaries, the ovarian tissue of the mice was lysed in the lysis buffer (Yeasen), and the ovarian lysate was incubated with CXCL10-bound glutathione agarose beads at 4°C for 4 h. The beads were washed with lysis buffer and washing buffer, then the antibody immunocomplex was sent to Biotech-Pack (Beijing, China) for mass spectrometry analysis of CXCL10-interacting proteins. The raw data generated by mass spectrometry were submitted to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) through the iProX partner repository and can be accessed using the dataset identifier PXD047305.

Cell culture, plasmid transfection, and co-immunoprecipitation

Mouse fibrosarcoma cells (MCA205) were obtained from the National Cancer Institute (Shanghai Yu Bo Biotech, Shanghai, China, Cat. No. YB867). Cells were cultured in RPMI-1640 with 10% FBS. MCA205 cells were transfected with pcDNA3.1(+)-IL18R1-EGFP-StrepII and pcDNA3.1(+)-CXCL10-TagRFP-Flag for immunoprecipitation. A total of 2 × 10⁶ cells were lysed in 250 µL IP buffer, and protein A/G beads (Yeasen) were pre-incubated at 4°C for 4 h to remove nonspecific binding. Next, 3 µg of mouse anti-Strep II-Tag monoclonal antibody or rabbit anti-DDDDK-tag (Flag) monoclonal antibody, along with rabbit or mouse control IgG, were coupled to the protein A/G beads in 250 µL IP buffer at 4°C for 4 h on a rotating wheel. Subsequently, the protein A/G-coupled control IgG or specific antibodies were incubated with pre-cleaned MCA205 cell lysates at 4°C overnight. Finally, after washed three times in IP buffer, protein blot analysis was conducted on the immunocomplexes bound to the anti-Strep II-Tag monoclonal antibody or rabbit anti-DDDDK-tag (Flag) monoclonal antibody.

Ovarian hematoxylin-eosin staining and follicle counting

To minimize the systematic errors caused by the estrus cycle, for all oocyte-related experiments and follicle counting, we used PMSG (5 IU per mice) to synchronize the cycle and promote follicle maturation. 48 h after PMSG injection, mice were sacrificed and the ovaries were harvested; one ovary was frozen in liquid nitrogen for protein sample preparation, and the other was washed with 0.9% NaCl solution, fixed with 4% paraformaldehyde (PFA) overnight, embedded in paraffin, and sectioned continuously at a thickness of 5 µm. The sections were deparaffinized in xylene, rehydrated gradually in a high to low concentration ethanol and distilled water, stained with hematoxylin and eosin, dehydrated in ethanol, cleared in xylene, and mounted with neutral resin (Solarbio, Beijing, China) for follicle counting under a microscope (Nikon Eclipse SI, Japan).

Every other section was counted because the distance between every three sections is approximately the size of an oocyte nucleus, and only visible and clearly identifiable follicles were included in the count. The follicle stages were classified according to the Pedersen criteria (19, 20). Primordial or primary follicles were surrounded by a single layer of flattened or cuboidal granulosa cells respectively, follicles that had multiple layers of cuboidal granulosa cells surrounding the oocyte were defined as secondary follicles, whereas follicles that had a clear cavity containing follicular fluid were defined as antral follicles (Fig. 1C).

Masson staining

Mouse ovaries were collected and washed with 0.9% NaCl solution, then soaked in 4% PFA (paraformaldehyde) and fixed overnight on a rotary shaker. The ovaries were embedded in paraffin and cut into 5 µm-thick sections. The sections were stained with the masson staining solution according to the manufacturer's protocol (SbjBio, Nanjing, China). In brief, the sections were dewaxed and incubated in Bouin's solution overnight at room temperature and were then stained with Aniline Blue and Scarlet to visualize collagen fibers and nuclei, respectively, under dark conditions at 4°C. After differentiation with Azure A, phosphomolybdic acid, and Fast Green, the sections were dehydrated, mounted, and observed under a microscope (Nikon Eclipse SI, Japan) after weak acid treatment.

Immunofluorescence and immunohistochemistry in paraffin sections

The paraffin sections were dewaxed and hydrated and then boiled in a citrate buffer for antigen retrieval. For immunohistochemistry, the sections were incubated in 3% hydrogen peroxide at room temperature for 15 min. The sections were circled with an immunohistochemical pen. After blocking with goat serum for 1 h, the paraffin sections were incubated with the primary antibody overnight at 4°C. For immunofluorescence, the next day, the secondary antibody and DAPI (4', 6-diamino-2-phenylpyridine) nuclear staining were applied to the paraffin sections, which were then mounted with an anti-quenching solution. For immunohistochemistry, the next day, the rabbit two-step detection kit (ZSGB-BIO, Beijing, China, Cat No. PV-9001) and Diaminobenzidine (DAB) chromogenic kit (ZSGB-BIO, Cat No. ZLI-9017) were employed according to the manufacturer’s instructions. The paraffin sections were counterstained with hematoxylin, dehydrated, cleared, and mounted with neutral resin (Absin Biotech, Shanghai, China, Cat No. abs9177). Fluorescence imaging was performed under a confocal microscope (Zeiss LSM 810, Germany or Nikon AXR, Japan), and immunohistochemistry imaging was performed under a microscope (Nikon Eclipse SI, Japan).

Oocyte collection and in-vitro maturation

For ovulated oocytes, mice were first injected with 5 IU PMSG (Pregnant Mare Serum Gonadotropin, Ningbo Second Hormone Factory (NSHF), Ningbo, China) I.P.; 48 h later, mice were injected with 5 IU HCG (Human Chorionic Gonadotropin, NSHF) to induce ovulation. 12 h later, mice were sacrificed and the ampulla of fallopian tube was torn open with two needles, the ovulated COCs (cumulus-oocyte complex) with maturated MII (metaphase II) oocytes were released into HEPES buffer.

For fully-grown GV (germ-vesicle) oocytes, ovaries were harvested as above, large antral follicles were punched with a syringe needle in HEPES (4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid) buffer and COCs were released; nude oocytes were separated from COCs by repeated blowing-and-suction with a mouth glass pipette (about 160 µm in diameter) in a MEM (Minimum Essential Medium) + medium (0.01 mM EDTA, 0.23 mM Na-pyruvate, 0.2 mM penicillin/streptomycin, and 3 mg/ml BSA in MEM, Thermo Fisher). Every 50 oocytes were cultured in 100 µL mini-drops of MEM + containing 20% FBS covered with mineral oil in an incubator with at 37.0°C, 5% O₂, 5% CO₂ in a humidified atmosphere. Before the experimental treatment, 2.5 µM milrinone was added to all media to inhibit the onset of meiosis.

Immunofluorescence staining of oocytes

After a brief rinse with PBS/0.05% PVP (polyvinylpyrrolidone, to prevent adhesion), oocytes were permeabilized with 0.5% Triton X-100/PHEM for 5 min, fixed in 3.7% PFA/PHEM (18.14 g Pipes ,6.5 g Hepes ,3.8 g EGTA ,0.99 g MgSO4,pH 7.0 w/ KOH) for 20 min, and washed three times (10 min each) with PBS/PVP. The oocytes were then incubated at room temperature in blocking buffer (1% BSA/PHEM, 100 mM glycine), followed by overnight incubation at 4°C with the primary antibody. After washed three times (10 min each) with PBST (PBS containing 0.05% Tween-20), the oocytes were incubated with a fluorescently labeled secondary antibody at room temperature for 45 min. The oocytes were stained with DAPI for 15 min, washed three times with PBST, and mounted on a glass slide with double-sided tape to secure the edges of the coverslip. Excess water was removed, an anti-quenching agent was added, and the coverslip was sealed with colorless nail polish around the edges. Imaging was performed under a confocal microscope (Zeiss LSM 810, Germany or Nikon AXR, Japan).

In vitro culture and treatment of mouse ovaries

Ovarian culture medium was α-MEM (Gibco, Cat No. C12571500BT) supplemented with 3 mg/mL BSA, 0.23 mM pyruvic acid, 50 µg/mL vitamin C, 0.03 U/mL FSH (Follicle Stimulating Hormone, Sigma, Cat No. F4021), 75 mg/L penicillin and 50 mg/L streptomycin. Gelatin sponges of an appropriate size were cut and placed in 24-well culture plates with about 100 µL culture medium. Mouse ovaries were obtained as above, separated, and cut into small pieces, then placed on the gelatin sponges. Ovary were treated 12 h. For Fig. 3P-S, the final conc. of purified recombinant CXCL10 and CXCL10 antibody were 1 µg/mL and 10 µg/mL respectively; for Fig. 6I-M, Fig. 7F-J, and Fig. 8G-M, the final conc. of purified recombinant CXCL10 and IL18R1 antibody were 1 µg/mL and 10 µg/mL respectively.

Image processing, measurement, and statistical analysis

The signal intensity in the original tif images (blot, DNA gel, immunofluorescence image, etc) were measured in image j. All statistical graphs showing western blots and DNA gels are based on three to six independent repetitions, follicle counting was based on five independent repetitions. Statistics on immunofluorescence image, masson staining, immunohistochemistry are based on three independent repetitions. Each dot on the graphs represents one data point. Detailed information about repeat times and number of data points are in figure legend. When the standard error of all individual data points in a group collected randomly was significantly smaller than the average value, the corresponding sample size was deemed appropriate and reliable. The data are presented as the mean ± standard error of the mean (SEM). For statistical comparison between two groups, we employed unpaired two-tailed t-test of GraphPad (Prism, San Diego, USA); for statistical comparison between three or more groups, we used ANOVA (one-way nonparametric analysis of variance, Prism). Statistical significance was determined at p-values less than 0.05.

WCV treatment protected ovarian function, whereas PD-1 antibody did not

Very few studies have investigated the impact of immunotherapy on the ovaries, and the present study addressed this research gap. We established a homograft tumor model in normal B6 mice with MCA205 cells (M group). This fibrosarcoma cell line is a malignant tumor line with rapid progression and a high death rate; it can form tumors in normal B6 mice in 1–2 weeks, making it an ideal model to study female fertility under various treatments.

After approximately 7 days of subcutaneous injection, MCA205 formed observable tumor nodes (Fig. 2B and 2C); these results are similar to those previously reported (21), indicating that the tumor model was successfully established. Next, we found that in the M group, the size and weight of ovaries as well as the follicle numbers at each stage were significantly decreased (Fig. 1A–D), indicating that cancer cells caused prominent damage to ovarian function. Moreover, Western blot analysis confirmed a decline in AMH (anti-Müllerian hormone) expression levels, indicating a decrease in female fertility (Fig. 1E and 1F). Furthermore, in the M group, the health status of ovarian cells significantly worsened, as indicated by a decreased PCNA (proliferating cell nuclear androgen) signal and an increased TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) signal (Fig. 1G–J).

Next, we employed two popular immunotherapies, anti-PD-1 monoclonal antibody (PM) and WCV (whole cancer vaccine, VM) therapy, and investigated whether they worsen or rescue the ovarian damage caused by cancer. We found that, as previously reported, both PD-1 and WCV injection significantly reduced tumor progression (Fig. 2A–D); however, they exhibited large differences in their impact on ovarian performance. The number of ovulated oocytes (mature metaphase II (MII) oocytes) in the M group was substantially lower than that in the control group; WCV treatment significantly increased this number in VM group, whereas PD-1 antibody treatment had no impact on the PM group mice (Fig. 2E and 2F). Second, the in vitro maturation rate (MII rate) of GV (germinal vesicle) oocytes retrieved from ovaries in the M group was significantly lower than that in the control group; WCV treatment significantly improved the MII rate in the VM group, whereas PD-1 antibody treatment had no impact on PM group mice (Fig. 2G and 2H). Third, the numbers of primordial follicles and developing follicles at each stage was significantly lower in the M group than in the control group; WCV treatment increased these numbers in the VM group, whereas PD-1 treatment did not have an obvious rescue effect on PM group mice (Fig. 2I–M).

CXCL10 was the primary factor for impaired ovarian function upon tumor invasion

Next, we explored the mechanism of the difference between the M, VM, and PM groups. Ovarian RNA sequencing revealed over 400 differentially expressed genes (DEGs) between the M group and the control group at the threshold of |log2(M/CTR)| ≥ 1. The DEGs profile of the WCV group was more similar to that of the control group, whereas the DEGs profile of the PD-1 group was different from that of both the control group and the VM group (Fig. 3A, supplementary Fig. 1A, and Supplementary dataset 1). KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis of the DEGs showed that many were involved in inflammation. (Fig. 3B).

Many studies have demonstrated that increased inflammation impairs ovarian function (22–24); therefore, we focused on inflammation-promoting cytokines next. We employed a microarray including 31 cytokines to enhance cytokine detection to detect the changes of these cytokines among control, M, VM, and PM group. Of these, the absolute conc. of CXCL10 was significantly higher than other cytokines, the most upregulated in the M group was CXCL10, and WCV treatment reduced the CXCL10 level close to the control in the VM group, whereas PD-1 antibody treatment had no impact on its expression in the PM group (Fig. 3C-I, supplementary Fig. 1B and supplementary Fig. 2, red dot-line rectangle, Supplementary dataset 2). We verified this change in both the plasma (Fig. 3J and 3K) and the ovaries (Fig. 3L and 3M). Subsequently, we used purified CXCL10 (Fig. 3O) to treat in vitro-cultured ovaries and found that CXCL10 significantly decreased proliferation (indicated by PCNA, Fig. 3P and 3Q) and increased DSBs (DNA double-strand breaks, indicated by γH2AX, Fig. 3R and 3S); anti-CXCL10 antibody treatment completely reversed these changes, suggesting that CXCL10 is a key cytokine impairing ovarian function.

To further investigate the details how Cxcl10 level was regulated, we did ovarian single-cell RNA sequencing analysis between Ctr, M, VM and PM group. Totally, 49553 transcriptomes were unsupervised clustering into 23 sub-clusters and annotated into 9 major cell types, namely granulosa cells (GCs), fibroblasts, theca cells, endothelial cells (ECs), ovarian surface epithelial cells (OSECs), oocytes, myeloid cells, T and NK cells, and erythrocytes (Fig. 4A and 4B). Noticeably, using gene signature scoring with Ucell, we found that scores of inflammatory response signature and apoptosis-related signature significantly increased in four major types of cells (granulosa cells, fibroblasts, theca cells, and endothelial cells) upon tumor invasion in M group, WCV treatment in VM group significantly reduced the scores close to the control, whereas PD-1 antibody treatment in PM group had no any rescue effects (Fig. 4C and 4D). Particularly, both the percentage of Cxcl10-expressing cells and the RNA level of Cxcl10 significantly increased upon tumor invasion in M group, WCV treatment in VM group significantly reduced the percentage and RNA level of Cxcl10 close to the control, whereas PD-1 antibody treatment in PM group had no any rescue effects (Fig. 4E). Besides, through CellChat interaction analysis, it was found that the number of cell types involved in the CXCL signaling pathway interactions significantly increased upon tumor invasion in the M group; WCV treatment in VM group significantly reduced the number close to the control, whereas PD-1 antibody treatment in PM group had less rescue effects (Fig. 4F).

IL18R1 is the dominant CXCL10 receptor in the ovaries

CXCR3 is a CXCL10 receptor expressed on CD8 + T cells (24), Treg cells (25), resident memory B cells (26), and tumor cells (27). CXCL10 binding to CXCR3 mediates the recruitment of CXCR3-expressing cells to the tumor or infected regions (28); however, the impact of CXCL10 on ovaries appeared to be different. Therefore, we hypothesized that another receptor dominating over CXCR3 might induce ovarian damage. We bound recombinant GST–CXCL10 onto glutathione agarose resin, incubated the resin with ovarian lysates, and then conducted mass spectroscopy. We characterized 61 proteins that may interact with CXCL10, among which IL18R1 was the only receptor (Fig. 5A, supplementary dataset 3). We verified the interaction between CXCL10 and IL18R1 through co-IP (co-immunoprecipitation) (Fig. 5B). Furthermore, we found that as an increasing amount of recombinant IL18R1 (Fig. 5C) was added, GST–CXCL10 bound to glutathione agarose resin pulled down increasing amounts of IL18R1 (Fig. 5D).

Interestingly, we did not bait CXCR3, the known CXCL10 receptor expressed by T cells, as the CXCL10-interacting protein in the ovaries. We examined the relative expression levels of CXCL10 and IL18R1 in the ovaries and tumors and found that the IL18R1:CXCR3 ratio in the ovaries was over 6-fold higher than that in the tumor, indicating that IL18R1 dominates over CXCR3 in binding to CXCL10 in the ovaries (Fig. 5E and 5F). In addition, we found that recombinant CXCL10–Strep II co-localized with IL18R1 on the membrane of both oocytes and GCs (granulosa cells) (Fig. 5G).

WCV treatment decreased tumor-induced fibrosis through the CXCL10→IL18R1→COL1A1 pathway

Fibrosis severely impairs ovarian function and is the main feature of reproductive aging (29) and polycystic ovary syndrome (30); in a previous study, antifibrosis drugs reinstated ovulation in mice that were reproductively old and obese by eradicating fibrotic collagen (31). IL18R1 was shown to promote COL1A1 expression through JNK (32). Therefore, we hypothesized that CXCL10 could induce fibrosis through IL18R1. Our results showed that tumor invasion significantly increased fibrosis in the M group; WCV treatment reduced fibrosis close to the control in the VM group, whereas PD-1 antibody treatment did not affect fibrosis in the PM group (Fig. 6A and 6B). Accordingly, the transcription levels of Col1a1 significantly increased in M group, WCV treatment reduced fibrosis close to the control in the VM group, whereas PD-1 antibody treatment did not have any rescue effects in PM group (Fig. 6C and 6D).

JNK and c-JUN, which regulate Col1a1 transcription in the ovaries, are downstream proteins of IL18R1 (32). COL1A1, p-c-JUN, and p-JNK were significantly elevated in the ovaries in the M group; WCV treatment reduced the levels of these proteins close to the control in the VM group, whereas PD-1 antibody treatment did not show any rescue effect on the levels of these proteins in the PM group (Fig. 6E–H).

Next, to further verify the connection between CXCL10 and IL18R1 in fibrosis induction, we compared recombinant CXCL10 treatment (CXCL10 group) and CXCL10 plus IL18R1 antibody treatment (10 + 18ab) in in vitro-cultured ovaries. Western blot analysis demonstrated that recombinant CXCL10 treatment sharply increased the levels of COL1A1, p-c-JUN, and p-JNK in the CXCL10 group, whereas IL18R1 antibody treatment reversed their levels close to the control in the 10 + 18ab group (Fig. 6I–K). Accordingly, masson staining showed that recombinant CXCL10 treatment sharply increased fibrosis in the CXCL10 group, whereas IL18R1 antibody treatment reversed their levels close to the control in the 10 + 18ab group (Fig. 6L and 6M).

These results indicate that in the ovaries, CXCL10 binds IL18R1 to induce downstream JNK→c-JUN activation, which subsequently promotes the transcription of COL1A1 and ovarian fibrosis (Fig. 6N).

WCV treatment decreased tumor-induced primordial follicle decrement through the CXCL10→IL18R1→AKT pathway

The decrease in primordial follicles (PMFs) is one of the main causes of reduced female fertility, and AKT overactivation is one of the main mechanisms for the decrease in PMFs (33). IL18R1 has also been shown to promote AKT activation (34). Therefore, we hypothesized that CXCL10 could induce a decrease in PMFs through IL18R1.

A previous study found that p-AKT can enter the nucleus and phosphorylate FOXO3A, which promotes the expression of genes necessary for PMF activation (35). Immunohistochemistry revealed that tumor invasion significantly increased nuclear p-FOXO3A in ovarian PMFs in the M group; WCV treatment reduced the level of nuclear p-FOXO3A close to the control level in the VM group, whereas PD-1 antibody treatment did not have any rescue effect on nuclear p-FOXO3A levels in the PM group (Fig. 7A and 7B). Similarly, Western blot analysis showed that tumor invasion significantly increased p-AKT and p-FOXO3A in the M group, and WCV treatment reduced their levels close to the control in the VM group, whereas PD-1 antibody treatment did not show any rescue effect in the PM group (Fig. 7C–E). However, we didn’t saw an increase of the ratio of growing follicles (PFs + SFs + AFs)/static follicles (PMFs), probably due to that tumor invasion significantly decreased the number of both PMFs and growing follicles (Supplementary Fig. 4 and Fig. 2I-M).

Next, to further verify the connection between CXCL10 and IL18R1 for PMF activation, we compared recombinant CXCL10 treatment and CXCL10 plus IL18R1 antibody treatment (10 + 18ab) in in-vitro cultured ovaries. Blot showed that recombinant CXCL10 treatment sharply increased the levels of p-AKT and p-FOXO3A in the CXCL10 group, while IL18R1 antibody treatment reversed their levels close to the control in the 10 + 18ab group (Fig. 7F-H). Accordingly, immunohistochemistry showed that recombinant CXCL10 treatment sharply increased the levels of p-FOXO3A in nucleus of PMFs from CXCL10 group, while IL18R1 antibody treatment reversed their levels close to the control in the 10 + 18ab group (Fig. 7I and 7J).

The upper results indicate that in the ovaries, CXCL10 can bind IL18R1 to induce downstream AKT→FOXO3A activation, which subsequently promotes the transcription of genes for PMF activation (Fig. 7K).

WCV treatment decreased tumor-induced inflammation through the CXCL10→IL18R1→NFκB pathway

Cancer tissues induce the production and release of various cytokines, many of which are pro-inflammatory (36, 37). Cytokines must bind corresponding membrane receptors that transduce pro-inflammatory signals (38). IL18R1 has been shown to activate p65, which then translocated to the nucleus to promote the transcription of pro-inflammatory genes (39); therefore, we hypothesized that CXCL10 could induce inflammation through IL18R1.

Western blot analysis demonstrated that tumor invasion significantly increased p-P65 and IFNγ levels in ovaries in the M group; WCV treatment reduced these levels close to the control in the VM group, whereas PD-1 antibody treatment did not show any rescue effect on these levels in the PM group (Fig. 8A–C). Furthermore, FACS and quantification (Supplementary Fig. 4, Fig. 8D-F) showed that in the M group, the percentage of M1 macrophages were significantly increased (34.97% in M, 27.75% in CTR), the percentage of M2 macrophages were significantly reduced (25.68% in M, 29.82% in CTR); WCV treatment significantly reduced the percentages of M1 macrophages (29.13% in WCV, meanwhile increased the percentage of M2 macrophages (26.47% in VM) close to the control in the VM group, whereas anti-PD-1 antibody treatment had no significant rescue effect in the PM group (M1, 33.85%; M2, 25.93%).

Next, to further verify the connection between CXCL10 and IL18R1 in the induction of inflammation, we compared recombinant CXCL10 treatment and CXCL10 plus IL18R1 antibody treatment in the in vitro-cultured ovaries. Western blot showed that recombinant CXCL10 treatment sharply increased the levels of p-p65 and IFNγ in the CXCL10 group, whereas IL18R1 antibody treatment reduced their levels close to the control in the 10 + 18ab group (Fig. 8G–I). Accordingly, we investigated the change in macrophage levels in the ovaries. Results showed that CXCL10 treatment significantly increased the level of the pro-inflammatory M1 macrophage marker INOS in the CXCL10 group, whereas CXCL10 plus IL18R1 antibody treatment reduced the INOS level close to the control in the 10 + 18ab group (Fig. 8J and 8K). On the other hand, CXCL10 treatment significantly decreased the level of the anti-inflammatory M2 macrophage marker CD206 in the CXCL10 group, whereas CXCL10 plus IL18R1 antibody treatment elevated the CD206 level close to the control in the 10 + 18ab group (Fig. 8L and 8M).

The above results indicated that in the ovaries, CXCL10 could bind IL18R1 to activate the downstream p-P65→IFNγ signal, which subsequently promoted the transformation of macrophages into pro-inflammatory type I macrophages (Fig. 8N).

CXCL10 antibody injection rescued the tumor-induced decrease in ovarian function

Next, we explored whether CXCL10 inhibition via CXCL10 antibodies could rescue ovarian function. The results showed that CXCL10 antibody injection significantly restored the number of follicles at each stage (Fig. 9A–E). Furthermore, we examined whether CXCL10 antibody injection could rescue the above ovarian abnormalities caused by tumor invasion.

First, CXCL10 antibody treatment in the CM group reversed the tumor-induced upregulation of p-JNK, p-c-JUN, and COL1A1 (Fig. 9F–I). In addition, CXCL10 antibody treatment in the CM group reversed the tumor-induced increase of ovarian fibrosis (Supplementary Fig. 5A and 5B). Second, CXCL10 antibody treatment in the CM group reversed the tumor-induced increase of p-AKT and p-FOXO3A levels (Fig. 9J–L). In addition, CXCL10 antibody treatment in the CM group could reverse the tumor-induced increase of nuclear p-FOXO3A in PMFs (Supplementary Fig. 5C and 5D). Third, CXCL10 antibody treatment in the CM group reversed the tumor-induced increase of p-P65 and IFNγ levels (Fig. 9M–O). Additionally, immunofluorescence showed that CXCL10 antibody treatment in the CM group significantly reversed the tumor-induced increase of pro-inflammatory M1 macrophage level (Fig. 9P and 9Q) and decrease of anti-inflammatory M2 macrophage level (Fig. 9R and 9S); Finally, FACS and quantification (Fig. 9T-V) showed that in the M group, the percentage of M1 macrophages were significantly increased (36.78% in M, 28.41% in CTR), the percentage of M2 macrophages were significantly reduced (24.15% in M, 29.95% in CTR); whereas anti-CXCL10 antibody treatment significantly reduced the percentages of M1 macrophages (29.85% in CM), meanwhile increased the percentage of M2 macrophages (29.89% in CM) close to the control in the CM group.

These results further indicated that CXCL10 is the key cytokine that induces ovarian damage and that CXCL10 antibodies can be used to prevent ovarian damage caused by tumor invasion.

Recombinant CXCL10–IL18R1 interface peptide injection rescued the tumor-induced decrease in ovarian function

The above results demonstrated that CXC10 antibody injection rescued ovarian damage; however, it could also inhibit the normal function of CXCL10 outside of the ovaries (28). Thus, specifically inhibiting the binding between CXCL10 and IL18R1 could rescue the ovarian damage without affecting its normal function in other tissues. We analyzed the interaction interface of CXCL10–IL18R1 with AlphaFold and selected the two models with the highest scores. In model 1, AAs 74–82 of CXCL10 appeared to closely match AAs 245–306 AA of IL18R1; in model 2, AAs 45–67 of CXCL10 appeared to closely match AAs 243–297 of IL18R1. We fused the Flag sequence (DYKDDDDK) and cell-penetrating peptide sequence TAT (CYGRKKRRQRRR) to each of the upper CXCL10 regions and named them CIBB-1 (CXCL10-IL18R1 Binding Blocker 1) and CIBB-2 (CXCL10-IL18R1 Binding Blocker 2) (Fig. 10A and 10B, Supplementary movie 1 and 2). We named the combination of these two sequences CIBB and expected that it could specifically block the binding between CXCL10 and IL18R1 (Fig. 10C).

We found that when in vitro-cultured oocytes were treated by recombinant CXCL10 only, some CXCL10 could be internalized into the oocyte cytoplasm; however, when the oocytes were co-treated with CXCL10 and CIBB, CXCL10 internalization significantly decreased, suggesting that CIBB effectively blocked the binding of CXCL10 to IL18R1 (Fig. 10D and 10E). In addition, we found that CIBB treatment in the CI-M (MCA205 tumor + CIBB) group significantly reversed the tumor-induced decrease of ovary weight (Fig. 10F and 10G).

Furthermore, we examined whether CIBB injection could rescue the upper ovarian abnormalities caused by tumor invasion. First, CIBB treatment in the CI-M group reversed the tumor-induced upregulation of p-JNK, p-c-JUN, and COL1A1 level (Fig. 10H–K). Additionally, CIBB treatment in the CI-M group reversed the tumor-induced increase of ovarian fibrosis (Supplementary Fig. 6A and 6B). Second, CIBB treatment in the CI-M group reversed the tumor-induced increase of p-AKT and p-FOXO3A level (Fig. 10L–N). Additionally, CIBB treatment in the CI-M group reversed the tumor-induced increase of nuclear p-FOXO3A in PMFs (Supplementary Fig. 6C and 6D). Third, CIBB treatment in the CI-M group reversed the tumor-induced increase of p-P65 and IFNγ level (Fig. 10O–Q). Moreover, CIBB treatment in the CI-M group reversed the tumor-induced increase of pro-inflammatory M1 macrophages (Fig. 10R and 10S) and decrease of anti-inflammatory M2 macrophages (Fig. 10T and 10U). Finally, FACS and quantification (Fig. 10V-X) showed that in the M group, the percentage of M1 macrophages were significantly increased (37.76% in M, 32.30% in CTR), the percentage of M2 macrophages were significantly reduced (18.81% in M, 28.24% in CTR); whereas CIBB treatment in the CI-M group significantly reduced the percentages of M1 macrophages (31.81% in CI-M), meanwhile increased the percentage of M2 macrophages (26.18% in CI-M) close to the control.

These results further indicated that CXCL10 binding to IL18R1 is the key mechanism that induces ovarian damage and that CIBB can be used to specifically prevent CXCL10-induced ovarian damage caused by tumor invasion.

To the best of our knowledge, this is the first study to find that WCV treatment could protect female fertility, providing a favorable immunotherapy choice for young females with cancer. We believe that our study has the following novelties.

First, we verified that WCV treatment has several critical advantages over PD-1 checkpoint inhibitor therapy for female fertility, including preserving the PMF reserve and improving oocyte quality. In addition, we confirmed the functionality of WCV treatment in the context of cancer invasion. Sherene Loi and Karla J. Hutt showed that PD-L1 inhibitor treatment significantly damaged ovarian function, including oocyte number and quality (40); Pauline C Xu showed that PD-1 blockade decreased primordial follicle reserve but didn’t affect short-term fertility (41). However, they did not examine the impact in the context of cancer invasion. In our study, PD-1 inhibitor treatment did not further decrease ovarian function in tumor-bearing mice, probably because of the dominant detrimental impacts of cancer invasion on ovarian function.

Second, we showed that CXCL10 is a primary pro-inflammation cytokine that has a detrimental impact on the ovaries. CXCL10 was previously shown to enhance the efficacy of cancer immunotherapy (42); from this point of view, it is a “beneficial” cytokine. However, in the ovaries, we found that it induced multiple detrimental impacts on the ovaries, including fibrosis, follicle overactivation, and inflammation. This suggests that certain cytokines might exert positive or negative impacts on different tissues.

Third, we characterized the primary receptor for CXCL10 in the ovaries, IL18R1, and showed that it dominates over CXCR3 in binding CXCL10. Although IL18 is a common ligand for IL18R1, in the current study, we did not find significant differences in IL18 levels between groups. This suggests that different cytokines can bind to a specific receptor under different conditions.

Fourth, we designed a peptide, CIBB, that specifically and effectively blocked the interaction between CXCL10 and IL18R1. In addition, we showed that CIBB effectively protects ovarian function from damage by cancer invasion.

In conclusion, this is the first study to show that WCV treatment achieves the dual function of suppressing tumor progression while protecting ovarian function. In addition, our results indicate that CXCL10→IL18R1 might be a critical target for protecting ovarian function, and CIBB could be employed independently to specifically protect ovarian damage by cancer invasion.

ACF

Animal core facility

AFs

antral follicles

AMH

Anti-mullerian hormone

ANOVA

Analysis of variance

BSA

Bovine serum albumin

CIBB

CXCL10-IL18R1 binding blocker

COCs

Cumulus-oocyte complex

co-IP

Co-Immunoprecipitation

CPP

cell-penetrating peptide

DAB

Diaminobenzidine

DAPI

4', 6-diamino-2-phenylpyridine

DEGs

Differentially expressed genes

DMSO

dimethyl sulfoxide

DSBs

DNA double-strand breaks

DTT

Dithiothreitol

ECs

Endothelial cells

FACS

Fluorescence-activated cell sorting

FBS

Fetal bovine serum

FDA

Food and drug administration

FSH

Follicle stimulating hormone

GCs

Granulosa cells

Germ-vesicle

HBSS

Hanks balanced salt solution

HCG

Human chorionic gonadotropin

HEPES

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

HTF

High-throughput fluidics

i.p.

intraperitoneally

i.v.

intravascularly

IACUC

Institutional animal care and use committee

IPTG

Isopropyl-β-d-thiogalactoside

KEGG

Kyoto encyclopedia of genes and genomes

LC-MS

Liquid chromatography-mass spectrometry

MEM

Minimum essential medium

MII

Metaphase II

MTA

Mitoxantrone

MTA

mitoxantrone

NJMU

Nanjing Medical University

OSECs

Ovarian surface epithelial cells

PBS

Phosphate-buffered saline

PCNA

Proliferating cell nuclear androgen

PFA

Paraformaldehyde

PFs

Primary follicles

PMFs

Primordial follicles

PMSF

Phenylmethylsulfonylfluoride

PMSG

Pregnant mare serum gonadotropin

PVP

Polyvinylpyrrolidone

s.c.

subcutaneous

SEM

Standard error of the mean

SFs

secondary follicles

SPF

Specific pathogen-free

TAT

trans-acting activator of transcription of HIV

TAT

Trans-activator

TUNEL

Terminal deoxynucleotidyl transferase dUTP nick-end labeling

WCV

Whole cancer cell vaccine

Acknowledgement

This work was financially supported by the General Program of the National Natural Science Foundation of China to Feng-Song Wang (Grant No: 81972641) and Dong Zhang (Grant No: 32070840), Major Projects in Provincial and National Union Construction of Henan Medical Science Research Plan to Cui-Lian Zhang (Grant No: SBGJ202001002), and the National Natural Science Foundation Joint Key Project to Yun-Xia Cao (Grant No: U20A20350).

Author contributions

Dong Zhang, Cui-Lian Zhang, Feng-Song Wang, and Yun-Xia Cao designed the research. Cong-Rong Li is the primary charger in most of the experiments, data collection & analysis and figure preparation, wherein Shi-Ya Xie, Shu-Ping Zhang, and Zhi-Xia Yang made substantial contribution. All others assisted in some of the experiments. Dong Zhang wrote the manuscript with the assistance of Cong-Rong Li; Cui-Lian Zhang, Feng-Song Wang, and Yun-Xia Cao proofread and gave advice. All authors read and approved the final manuscript.

Availability of data and material

The data that support the findings of this study are available from the corresponding author upon reasonable request. Supplementary datasets 1-3 have also been deposited into Zenodo (https://zenodo.org/records/10806702), the DOI is 10.5281/zenodo.10806701. The accession code for RNA-seq (in Fig. 3A) submitted to NCBI Sequence Read Archive database was GSE248829, the accession code for the mass spectrometry raw data (in Fig. 4A) submitted to the ProteomeXchange Consortium was PXD047305.

COMPETING INTERESTS

The authors declared that they have no competing interests to this work.

Hegde PS, Chen DS. Top 10 Challenges in Cancer Immunotherapy. Immunity. 2020 Jan 14;52(1):17-35.
Waldman AD, Fritz JM, Lenardo MJ. A guide to cancer immunotherapy: from T cell basic science to clinical practice. Nat Rev Immunol. 2020 Nov;20(11):651-668.
Kennedy LB, Salama AKS. A review of cancer immunotherapy toxicity. CA Cancer J Clin. 2020 Mar;70(2):86-104.
Spitzer MH, Carmi Y, Reticker-Flynn NE, Kwek SS, Madhireddy D, Martins MM, Gherardini PF, Prestwood TR, Chabon J, Bendall SC, Fong L, Nolan GP, Engleman EG. Systemic Immunity Is Required for Effective Cancer Immunotherapy. Cell. 2017 Jan 26;168(3):487-502.e15.
Sanmamed MF, Chen L. A Paradigm Shift in Cancer Immunotherapy: From Enhancement to Normalization. Cell. 2018 Oct 4;175(2):313-326.
Carlino MS, Long GV. Ipilimumab Combined with Nivolumab: A Standard of Care for the Treatment of Advanced Melanoma? Clin Cancer Res. 2016 Aug 15;22(16):3992-8.
O'Connor JM, Fessele KL, Steiner J, Seidl-Rathkopf K, Carson KR, Nussbaum NC, Yin ES, Adelson KB, Presley CJ, Chiang AC, Ross JS, Abernethy AP, Gross CP. Speed of Adoption of Immune Checkpoint Inhibitors of Programmed Cell Death 1 Protein and Comparison of Patient Ages in Clinical Practice vs Pivotal Clinical Trials. JAMA Oncol. 2018 Aug 1;4(8):e180798.
Vardhana S, Cicero K, Velez MJ, Moskowitz CH. Strategies for Recognizing and Managing Immune-Mediated Adverse Events in the Treatment of Hodgkin Lymphoma with Checkpoint Inhibitors. Oncologist. 2019 Jan;24(1):86-95.
Johnson DE, Burtness B, Leemans CR, Lui VWY, Bauman JE, Grandis JR. Head and neck squamous cell carcinoma. Nat Rev Dis Primers. 2020 Nov 26;6(1):92.
Hsu FS, Su CH, Huang KH. A Comprehensive Review of US FDA-Approved Immune Checkpoint Inhibitors in Urothelial Carcinoma. J Immunol Res. 2017;2017:6940546.
Akinleye A, Rasool Z. Immune checkpoint inhibitors of PD-L1 as cancer therapeutics. J Hematol Oncol. 2019 Sep 5;12(1):92.
Teo MY, Rosenberg JE. Refining existing knowledge and management of bladder cancer. Nat Rev Urol. 2019 Feb;16(2):75-76.
Gracia CR, Jeruss JS. Lives in the balance: women with cancer and the right to fertility care. J Clin Oncol. 2013 Feb 20;31(6):668-9.
Donnez J, Dolmans MM. Fertility Preservation in Women. N Engl J Med. 2018 Jan 25;378(4):400-401.
Menzer C, Beedgen B, Rom J, Duffert CM, Volckmar AL, Sedlaczek O, Richtig E, Enk A, Jäger D, Hassel JC. Immunotherapy with ipilimumab plus nivolumab in a stage IV melanoma patient during pregnancy. Eur J Cancer. 2018 Nov;104:239-242.
Bolze PA, You B, Lotz JP, Massardier J, Gladieff L, Joly F, Hajri T, Maucort-Boulch D, Bin S, Roux A, Rousset P, Villeneuve L, Alves-Ferreira M, Grazziotin-Soares D, Mercier C, Freyer G, Golfier F. Successful pregnancy in a cancer patient previously cured of a gestational trophoblastic tumor by immunotherapy. Ann Oncol. 2020 Jun;31(6):823-825.
Azim HA Jr, Pavlidis N, Peccatori FA. Treatment of the pregnant mother with cancer: a systematic review on the use of cytotoxic, endocrine, targeted agents and immunotherapy during pregnancy. Part II: Hematological tumors. Cancer Treat Rev. 2010 Apr;36(2):110-21.
Yang H, Xia L, Chen J, Zhang S, Martin V, Li Q, Lin S, Chen J, Calmette J, Lu M, Fu L, Yang J, Pan Z, Yu K, He J, Morand E, Schlecht-Louf G, Krzysiek R, Zitvogel L, Kang B, Zhang Z, Leader A, Zhou P, Lanfumey L, Shi M, Kroemer G, Ma Y. Stress-glucocorticoid-TSC22D3 axis compromises therapy-induced antitumor immunity. Nat Med. 2019 Sep;25(9):1428-1441.
Pedersen T, Peters H. Proposal for a classification of oocytes and follicles in the mouse ovary. J Reprod Fertil. 1968 Dec;17(3):555-7.
Sonigo C, Jankowski S, Yoo O, Trassard O, Bousquet N, Grynberg M, Beau I, Binart N. High-throughput ovarian follicle counting by an innovative deep learning approach. Sci Rep. 2018 Sep 10;8(1):13499.
Huang N, Liu D, Lian Y, Chi H, Qiao J. Immunological Microenvironment Alterations in Follicles of Patients With Autoimmune Thyroiditis. Front Immunol. 2021 Nov 17;12:770852.
Jiao X, Zhang X, Li N, Zhang D, Zhao S, Dang Y, Zanvit P, Jin W, Chen ZJ, Chen W, Qin Y. Treg deficiency-mediated TH 1 response causes human premature ovarian insufficiency through apoptosis and steroidogenesis dysfunction of granulosa cells. Clin Transl Med. 2021 Jun;11(6):e448.
Navarro-Pando JM, Alcocer-Gómez E, Castejón-Vega B, Navarro-Villarán E, Condés-Hervás M, Mundi-Roldan M, Muntané J, Pérez-Pulido AJ, Bullon P, Wang C, Hoffman HM, Sanz A, Mbalaviele G, Ryffel B, Cordero MD. Inhibition of the NLRP3 inflammasome prevents ovarian aging. Sci Adv. 2021 Jan 1;7(1):eabc7409.
Ozga AJ, Chow MT, Lopes ME, Servis RL, Di Pilato M, Dehio P, Lian J, Mempel TR, Luster AD. CXCL10 chemokine regulates heterogeneity of the CD8+ T cell response and viral set point during chronic infection. Immunity. 2022 Jan 11;55(1):82-97.e8.
Zhang Y, Zuo C, Liu L, Hu Y, Yang B, Qiu S, Li Y, Cao D, Ju Z, Ge J, Wang Q, Wang T, Bai L, Yang Y, Li G, Shao Z, Gao Y, Li Y, Bian R, Miao H, Li L, Li X, Jiang C, Yan S, Wang Z, Wang Z, Cui X, Huang W, Xiang D, Wang C, Li Q, Wu X, Gong W, Liu Y, Shao R, Liu F, Li M, Chen L, Liu Y. Single-cell RNA-sequencing atlas reveals an MDK-dependent immunosuppressive environment in ErbB pathway-mutated gallbladder cancer. J Hepatol. 2021 Nov;75(5):1128-1141.
MacLean AJ, Richmond N, Koneva L, Attar M, Medina CAP, Thornton EE, Gomes AC, El-Turabi A, Bachmann MF, Rijal P, Tan TK, Townsend A, Sansom SN, Bannard O, Arnon TI. Secondary influenza challenge triggers resident memory B cell migration and rapid relocation to boost antibody secretion at infected sites. Immunity. 2022 Apr 12;55(4):718-733.e8.
Hirth M, Gandla J, Höper C, Gaida MM, Agarwal N, Simonetti M, Demir A, Xie Y, Weiss C, Michalski CW, Hackert T, Ebert MP, Kuner R. CXCL10 and CCL21 Promote Migration of Pancreatic Cancer Cells Toward Sensory Neurons and Neural Remodeling in Tumors in Mice, Associated With Pain in Patients. Gastroenterology. 2020 Aug;159(2):665-681.e13.
Moreno Ayala MA, Campbell TF, Zhang C, Dahan N, Bockman A, Prakash V, Feng L, Sher T, DuPage M. CXCR3 expression in regulatory T cells drives interactions with type I dendritic cells in tumors to restrict CD8+ T cell antitumor immunity. Immunity. 2023 Jul 11;56(7):1613-1630.e5.
Secomandi L, Borghesan M, Velarde M, Demaria M. The role of cellular senescence in female reproductive aging and the potential for senotherapeutic interventions. Hum Reprod Update. 2022 Feb 28;28(2):172-189.
Zhou Y, Lan H, Dong Z, Li W, Qian B, Zeng Z, He W, Song JL. Rhamnocitrin Attenuates Ovarian Fibrosis in Rats with Letrozole-Induced Experimental Polycystic Ovary Syndrome. Oxid Med Cell Longev. 2022 May 26;2022:5558599.
Umehara T, Winstanley YE, Andreas E, Morimoto A, Williams EJ, Smith KM, Carroll J, Febbraio MA, Shimada M, Russell DL, Robker RL. Female reproductive life span is extended by targeted removal of fibrotic collagen from the mouse ovary. Sci Adv. 2022 Jun 17;8(24):eabn4564.
Wang C, Sun Y. Induction of Collagen I by CXCL10 in Ovarian Theca-Stroma Cells via the JNK Pathway. Front Endocrinol (Lausanne). 2022 Apr 1;13:823740.
Jang H, Lee OH, Lee Y, Yoon H, Chang EM, Park M, Lee JW, Hong K, Kim JO, Kim NK, Ko JJ, Lee DR, Yoon TK, Lee WS, Choi Y. Melatonin prevents cisplatin-induced primordial follicle loss via suppression of PTEN/AKT/FOXO3a pathway activation in the mouse ovary. J Pineal Res. 2016 Apr;60(3):336-47.
Sahar S, Dwarakanath RS, Reddy MA, Lanting L, Todorov I, Natarajan R. Angiotensin II enhances interleukin-18 mediated inflammatory gene expression in vascular smooth muscle cells: a novel cross-talk in the pathogenesis of atherosclerosis. Circ Res. 2005 May 27;96(10):1064-71.
Han L, Huang Y, Li B, Wang W, Sun YL, Zhang X, Zhang W, Liu S, Zhou W, Xia W, Zhang M. The metallic compound promotes primordial follicle activation and ameliorates fertility deficits in aged mice. Theranostics. 2023 May 21;13(10):3131-3148.
Jang JH, Kim DH, Surh YJ. Dynamic roles of inflammasomes in inflammatory tumor microenvironment. NPJ Precis Oncol. 2021 Mar 8;5(1):18.
Greten FR, Grivennikov SI. Inflammation and Cancer: Triggers, Mechanisms, and Consequences. Immunity. 2019 Jul 16;51(1):27-41.
Saxton RA, Glassman CR, Garcia KC. Emerging principles of cytokine pharmacology and therapeutics. Nat Rev Drug Discov. 2023 Jan;22(1):21-37.
Kanakaraj P, Ngo K, Wu Y, Angulo A, Ghazal P, Harris CA, Siekierka JJ, Peterson PA, Fung-Leung WP. Defective interleukin (IL)-18-mediated natural killer and T helper cell type 1 responses in IL-1 receptor-associated kinase (IRAK)-deficient mice. J Exp Med. 1999 Apr 5;189(7):1129-38.
Winship AL, Alesi LR, Sant S, Stringer JM, Cantavenera A, Hegarty T, Requesens CL, Liew SH, Sarma U, Griffiths MJ, Zerafa N, Fox SB, Brown E, Caramia F, Zareie P, La Gruta NL, Phillips KA, Strasser A, Loi S, Hutt KJ. Checkpoint inhibitor immunotherapy diminishes oocyte number and quality in mice. Nat Cancer. 2022 Aug;3(8):1-13.
Xu PC, Luan Y, Yu SY, Xu J, Coulter DW, Kim SY. Effects of PD-1 blockade on ovarian follicles in a prepubertal female mouse. J Endocrinol. 2021 Nov 24;252(1):15-30.
Xu G, Liu K, Chen X, Lin Y, Yu C, Nie X, He W, Karin N, Luan Y. Hydrogel-mediated tumor T cell infiltration and immune evasion to reinforce cancer immunotherapy. Nanoscale Horiz. 2024 Jan 29;9(2):295-304.

There is NO Competing Interest.

Supplementarydataset1.xlsx
Supplementary Data Set 1
Supplementarydataset2.xlsx
Supplementary Data Set 2
Supplementarydataset3.xlsx
Supplementary Data Set 3
SuppFig..pdf
SupplementaryData.docx
Supplementarymovie1.avi
Supplementary movie 1 Related to Fig. 9A. This file is model 1 showing the interface contact between CXCL10 and IL18R1. In the magnified interface region, CXCL10 sequence is highlighted in red, IL18R1 sequence is highlighted in green.
Supplementarymovie2.avi
Supplementary movie 2 Related to Fig. 9B. This file is model 2 showing the interface contact between CXCL10 and IL18R1. In the magnified interface region, CXCL10 sequence is highlighted in purple, IL18R1 sequence is highlighted in green.

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Whole Cancer Cell Vaccine Protects Ovarian Functions in Tumor-bearing Mice through Downregulating CXCL10

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Abstract

Figures

Introduction

Material and Methods

Antibodies

Mice Model

RNA sequencing and analysis

Multiplex immunoassay

Tissue dissociation and single-cell suspension preparation

Single-cell RNA sequencing and data analysis

Flow cytometric analysis

Expression and purification of IL18R1 in SF9 cells

Expression and purification of CXCL10 in Ecoli, GST-pulldown, and mass spectrometry

Cell culture, plasmid transfection, and co-immunoprecipitation

Ovarian hematoxylin-eosin staining and follicle counting

Masson staining

Immunofluorescence and immunohistochemistry in paraffin sections

Oocyte collection and in-vitro maturation

Immunofluorescence staining of oocytes

In vitro culture and treatment of mouse ovaries

Image processing, measurement, and statistical analysis

Results

WCV treatment protected ovarian function, whereas PD-1 antibody did not

CXCL10 was the primary factor for impaired ovarian function upon tumor invasion

IL18R1 is the dominant CXCL10 receptor in the ovaries

WCV treatment decreased tumor-induced fibrosis through the CXCL10→IL18R1→COL1A1 pathway

WCV treatment decreased tumor-induced primordial follicle decrement through the CXCL10→IL18R1→AKT pathway

WCV treatment decreased tumor-induced inflammation through the CXCL10→IL18R1→NFκB pathway

CXCL10 antibody injection rescued the tumor-induced decrease in ovarian function

Recombinant CXCL10–IL18R1 interface peptide injection rescued the tumor-induced decrease in ovarian function

Discussion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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