CD1d Modulation Influences Tumor Immune Crosstalk in Triple-Negative Breast Cancer (TNBC) through NKT and Macrophage Interaction 2257312

Abstract

Abstract Introduction TNBC remains an aggressive malignancy with immunosuppressive microenvironment. The lipid antigen-presenting molecule CD1d, a key regulator of Natural Killer T (NKT) cell activation, was found to be overexpressed in TNBC and can be a useful therapeutic target. Methods TNBC cells (MDAMB231 and MDAMB468) and monocytes (THP-1) were treated with CD1d modulators Zerumbone (ZER), α-Galactosylceramide (AGC), and Thymosin Alpha 1 (TA). We employed Immunofluorescence, flow cytometry and western blotting to assess CD1d. Cell adhesion, proliferation assays were performed using the RTCA method. For the NKT cell isolation from human PBMCs, we have used NKT isolation kit. Coculture assays were performed with NKT cells and TNBC cells as well as THP-1. The ELISA technique was used to assess cytokine secretion and correlated with NKT activation. Results Our findings identified differential CD1d isoform expression between the two TNBC cell lines and demonstrated that CD1d modulators ZER, AGC, and TA significantly alter tumor proliferation, migration, and angiogenesis in vitro. We compared CD1d expression in TNBC cell lines, revealing differential CD1d (37 kDa) and (48 kDa) isotypes. CD1d siRNA knockdown in both cell lines reduced these oncogenic phenotypes (proliferation, EMT and angiogenesis), confirming CD1d’s tumor-intrinsic effects. To further investigate the immunological context of CD1d modulation, co-culture of TNBC cells with human NKT cells and macrophages (THP-1 derived). CD1d upregulation by AGC and TA enhanced IFN-γ release in NKT co-cultures with TNBC cells, whereas ZER reduced IFN-γ secretion. Conclusion CD1d modulation differentially regulates TNBC immune interactions. ZER acts as a CD1d antagonist and may be beneficial in immunosuppressive TNBC with low NKT activity. In contrast, AGC and TA can be useful in killing TNBC cells through NKT mediated cytotoxicity through CD1d upregulation. CD1d modulators may therefore serve as novel therapeutic agents to reshape the TNBC immune microenvironment. Funding Source n/a Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)