Blocking the CXCL10-IL18R1 Axis Protects Ovaries from Tumor-induced Damage

Description

The ovary is susceptible to harmful environmental factors (hEFs). Tumor invasion is an intracorporal hEF. However, it is unclear whether and how tumor invasion compromises ovarian function, making it challenging to target and prevent this harm prior to diagnosis and cancer treatment. To develop a targeted therapy to protect ovarian function during tumor invasion, we used MCA205 (mouse fibrosarcoma) cell-allotransplanted B6 mice as a basic model (M group), and two popular therapeutic models—PD-1 monoclonal antibody injection in allotransplanted B6 mice (PD-1 group) and whole cancer cell vaccine (WCV) injection in allotransplanted B6 mice (WCV group)—to facilitate the discovery process. As expected, tumor infiltration in the M group reduced ovarian function in multiple ways. Interestingly, WCV injection strongly restored these anomalies, whereas PD-1 did not. Next, plasma cytokine microarrays revealed that the level of CXCL10 increased in the M group and was the best rescue in the WCV group. Next, we baited the sole CXCL10 receptor found in the ovaries, IL18R1. CXCL10 impairs ovarian function via three pathways: generating fibrosis through CXCL10→IL18R1→p-JNK→COL1A1, encouraging primordial follicle overactivation through CXCL10→IL18R1→p-AKT, and increasing ovarian inflammation through CXCL10→IL18R1→p-P65. To improve ovarian function in the M group, we blocked the CXCL10→IL18R1 pathway with CXCL10 antibody or CIBB, an interface peptide. This study provides mechanical evidence and translational evidence for how tumor invasion compromises ovaries functioning and how to target and protect ovaries during tumor invasion. Supplementary dataset 1 Related to Fig. 3A and 3B. This file includes two sheets. “All_samples-fpkm” includes FPKM values of all identified genes in ovaries from the CTR, M, VM, and PM groups (three repeats per group); “heatmap” includes all average log2 values (from three repeats per group) of 435 DEGs (differentially expressed genes) at the threshold of |log2(M/CTR)| ≥ 1. Supplementary dataset 2 Related to Fig. 3C. This file includes 33 sheets. “Summary” includes all conc. Values (pg/mL) of 31 plasma DECs (differentially expressed cytokines); “Curves” include standard curves for all 31 cytokines; each of the other 31 sheets includes detailed assays for each plasma cytokine for the CTR, M, VM, and PM groups (five repeats per group). Supplementary dataset 3 Related to Fig. 5A. This file includes two sheets. “peptides” includes all related values of all identified peptides; “Protein Groups” includes the related info. All the proteins corresponded to the identified peptides.

Steps to reproduce

RNA sequencing and analysis RNA samples were obtained from the ovaries of the mice. Isolating RNA, high-throughput sequencing, and data analysis were done 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 data in Fig. 3A, the accession number was GSE248829. Genes whose absolute value of the log2 (treated/control) ratio was greater than or equal to 1.2 and whose q value was less than 0.001 were considered differentially expressed genes (DEGs). Multiplex immunoassay Multiplex immunoassays were performed 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 replicates per group). Fifty microlitres of the collected sample were subjected to cytokine measurement via 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 via Bio-Plex Manager software version 6.1 (Bio-RAD). Mass spec indentification of CXCL10-interacting proteins To identify CXCL10-interacting proteins in the ovaries, the ovarian tissue of the mice was lysed in 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, and the antibody immunocomplex was subsequently 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 via the dataset identifier PXD047305.

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