(818) Distinct effects of different programmed cell death modalities on immune microenvironment of major molecular subtypes of breast cancer

Introduction/Rationale: Programmed cell death (PCD) is now classified as immunogenic or non-immunogenic based on its ability to trigger immune responses. The three major PCD types are apoptosis, autophagic cell death (ACD), and necroptosis. Immune cells like macrophages and lymphocytes play a vital role in tumour microenvironment. Induction of cell death is a desirable end point for anti-cancer therapies. However, they may sometimes result in immunosuppressive microenvironment with altered immune cell properties that favour the tumour progression or relapse.

Methods: MCF-7 (hormone receptors⁺) and MDA-MB-231 (triple negative) BC cells were treated with paclitaxel, shikonin and metformin to induce apoptosis, necroptosis, and ACD respectively. The conditioned medium (CM) was collected and JURKAT (T lymphocyte) and THP-1 (macrophage) cells were incubated with it. Post-incubation, RT-qPCR was used to assess lymphocyte (Th1, Th2, Th17, Th22, and Treg) and macrophage polarization (M1, M2) markers in the above cell types. Functional assays including cell cycle analysis and differentiation and phagocytosis assessment (flow cytometry and confocal microscopy) were carried out to gauze phenotypic effects on the immune cells. Further, PD-1/PD-L1 expression was quantified at transcriptional and translation level on lymphocytes and cancer cells.

Results: Paclitaxel (1 µM), metformin (2.5, 5mM), and shikonin (2.5 µM) induced cell death in MCF-7 and MDA-MB-231 cells. CM significantly (P-value < 0.005) changed the expression of IL-1β, STAT-5, and iNOS (M1 phenotype) and IL-4, GATA-3, and IFN-Ύ (Th1, Th2 subtype). RT-qPCR revealed drug-specific shifts in T lymphocyte and macrophage molecular and phenotypic properties, indicating distinct immuno-modulatory effects.

Conclusion: PCD-induced breast cancer cell supernatants modulate immune cell behavior, showing drug-specific effects that can guide therapies to enhance anti-tumor immunity and prevent immune suppression.