Journal: bioRxiv

Article Title: IL1β/IL1R1/IRAK4 Drives Inflammatory Ovarian Cancer Seeding at the inflamed sites and Is Reversed by an IRAK4 inhibitor UR241-2

doi: 10.64898/2026.04.30.722105

Figure Lengend Snippet: ( A-B ): Analysis of microarray data (TCGA-381-tpm-gencode36) of ovarian cancer patients using R2 Genomics Analysis and Visualization Platform tools showed that IL1β and IL1R1 mRNA overexpression predicts poor survival. ( C ): Schema of testing the role of Il1β, Il1r1 and Il1rn (Il1r1 antagonist) in ovarian cancer burden both at the needle injury and omental sites. ( D-E ): HGS-3 murine high-grade serous EOC cells (4.5 million/per mice) were implanted intraperitoneally using 21 sterile gauge needle in C57BL/6 WT and C57BL/6 Il1r1 KO mice. Mice were observed for 45-50 days and euthanized. Tumors formed on needle injury site both protruding at the skin, and, in the peritoneum, and on the omentum, shown by red arrows, were isolated, weighed and frozen in liquid nitrogen. Lavages via washing with sterile PBS(5mL) were also collected. The studies were repeated thrice. A representative experiment is shown. ( F ): Weights of the omental did not differ between C57BL/6 wt and Il1r1 KO mice. ( G ): Weights of the peritoneal/skin tumors differed significantly between C57BL/6 wt and Il1r1 KO mice groups. * indicates <0.05. ( H-K ): Tumors formed at the needle injury site in C57BL/6 wt and Il1rn KO mice are pointed with the red arrows. ( I -upper vs I -lower): Images of the omental and peritoneal /skin tumors harvested from the euthanized Il1r1 KO mice are shown. ( J ): Omental tumor weights in C57BL/6 wt did not differ between Il1rn k ° mice groups. ( L -upper vs L -lower): Images of the omental and peritoneal /skin tumors harvested from the euthanized Il1rn KO mice are shown. ( M ): Weights of the tumors formed on the needle injury site in C57BL/6 WT mice did not differ for Il1rn KO mice groups. This experiment was repeated twice. Tumor sizes were analyzed via non-parametric T-test using Graph-Prism version -7 or higher. * indicates <0.05. ( N ): Il1r1 KO does not significantly impact myeloid percentages in the peritoneal lavage and tumor. A) Peritoneal lavage and tumor percentages and cell numbers of CD11b+Ly6G+ neutrophils from flow cytometry data of wild type and IL1R1KO mice injected with HGS3 tumors. B) Peritoneal lavage percentages and cell numbers of F4/80loMHCIIhi and F4/80hiMHCIILow macrophages from the peritoneal lavage and percentage of MHCII hi and MHCII Low macrophages from tumors from flow cytometry data of wild type and IL1R1 KO mice injected with HGS3 tumors. N = 15-20 mice per group from 3 independent experiments. Statistical significance was determined using Mann-Whitney test, *p < 0.033, **p < 0.002, ***p < 0.001. ( O ): Table shows that none of the IL1β, IL1R1 and myd88/TLR targeted agents has been approved for treatment of a malignancy yet. ( P ): Schema of IL1β/IRAK4 signaling pathway. Scheme shows that inhibiting IRAK4 can centrally block IL1/TLR driven signaling in ovarian malignancies. ( Q ): Table summarizes the status of 3 IRAK4 inhibitors undergoing clinical trials. ( R ): Using GENT2 database we observed that compared to normal ovaries, malignant ovaries overexpress IRAK4 mRNA. ( S ): GENT2 database also showed that higher stages of EOC disease significantly increased IRAK4 mRNA expression. Analysis of the EOC patient’s microarray data using GENT2 tools showed that IRAK4 mRNA expression was altered in various stages of disease. Two-sample T-test showed statistical differentiations: IA vs I (p=0.004); IA vs II (p=0.007); IA vs III (p<0.001); IC vs IIA (p=0.002); IC vs III (p<0.001); IIA vs I (p<0.001); IIA vs II (p<0.001); IIA vs III (p<0.001); IIA vs IIC (p=0.006); IIA vs IIIC (p=0.001); IIC vs III (p<0.001); III vs IIB (p=0.004); IIIA vs I (p=0.001); IIIA vs II (p=0.004); IIIA vs III (p<0.001); IIB vs I (p=0.008); IIIB vs III (p=<0.001); IV vs I (p=0.004); IV vs IIA (p=0.005); IV vs III (p=<0.001). ( http://gent2.appex.kr/gent2/ , date accessed 10/2/2023). ( T-U ): Analysis of microarray data (TCGA-381-tpm-gencode36) of ovarian cancer patients using R2 Genomics Analysis and Visualization Platform tools showed that IRAK4 and IRAK1 mRNA overexpression predicts poor survival in EOC patients.

Article Snippet: Il1r1 KO (B6.129S7-Il1r1tm1Imx/J), Il1rn KO ( ) (B6.129S-Il1rn tm1Dih/J) ( ) and Nlrp3 KO (Strain #:021302 - B6.129S6-Nlrp3tm1Bhk/J) mice were bred in-house from the breeders obtained from Jax laboratories.

Techniques: Microarray, Over Expression, Sterility, Isolation, Flow Cytometry, Injection, MANN-WHITNEY, Blocking Assay, Clinical Proteomics, Expressing