Abstract 5769: Silibinin profiles the immune microenvironment via mast cell regulation: Potential immunomodulation in cancer prevention and therapy

Abstract Immune cells are critical check points, not only for foreign pathogens, but also for proper growth and proliferation of bodies’ own cells. They play crucial and complex roles in cancer, beginning at initiation of malignancy, proliferation of tumors, establishment of the cancer microenvironment, to metastasis and spread of malignant cells throughout the body. In our ongoing studies with silibinin (SB; flavonolignan obtained from milk thistle seeds), we showed that it is an effective preventive natural agent for several cancers including, basal cell carcinoma, prostrate cancer, colon cancer, and hepatocellular carcinoma. SB was also shown to regulate immune responses in several of these studies. Notably, SB treatment was found to significantly regulate mast cell (MC) infiltration and functioning in our completed and ongoing studies. However, since MCs are associated with the functions of other immune cells, it is also important to delve deeply into the downstream effects of SB treatment on other immune components regulated directly and/or indirectly by MCs. Thus, in the present study, in-depth mechanistic aspects of SB treatment on other cells of the tumor immunological microenvironment were determined, leveraging SB regulation of MCs. MCs were derived from bone marrow (BMMCs) of C57BL/6 mice in presence of IL-3 and SCF and confirmed using flow cytometry (dual staining: cKit/Fc€RI). Thereafter, BMMCs were exposed to 25-100 µM SB and two concentrations (25 µM; SB 25 and 100 µM; SB 100) were selected for further experiments. BMMCs treated with SB 25, SB 100, and vehicle control were subjected to proteomics (LCMS on Fusion Lumos mass spectrometer); 3575 proteins were identified using the annotated mouse proteome. Statistical analysis was performed using ANOVA followed by Fisher’s posthoc analysis (p<0.05, FDR<0.01) and 166 proteins were found to have statistically significant differential expression amongst the three groups (n=4/group). These 166 proteins were selected for further pathway analysis using Ingenuity Pathway Analysis (IPA; Qiagen). Stringent IPA filter settings were used to select pathways specifically “demonstrated experimentally” in “immune cells/cell lines” for further analysis. Top canonical pathways associated with SB treatment of immune cell regulation via MCs were found to be eukaryotic RNA processing (processing of capped intro-containing pre-mRNA), ISG15 mediated immune responses (ISG15 antiviral mechanism), eukaryotic-translation elongation, and RHO GTPase pathway. Important nodes determined were TNF, CD-28, IL-2, IL-4, IL-15, NR4A1, IFNB, and RAC2, suggesting SB plays an important immunomodulatory role via regulation of MCs. Further studies are warranted to delineate these mechanisms in cancers as well as other injury mechanisms and SB’ therapeutic potential (supported by R01 CA140368). Citation Format: Neha Mishra, Sandeep Paudel, Chapla Agarwal, Rajesh Agarwal. Silibinin profiles the immune microenvironment via mast cell regulation: Potential immunomodulation in cancer prevention and therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5769.