<p>Introduction: Pancreatic Ductal Adenocarcinoma (PDAC) is one of the most malignant gastrointestinal tumors. M1 macrophage, a subtype within the Tumor Microenvironment (TME), plays a vital role in the development of cancer. Despite its anti-tumoral functions, the specific mechanisms of its action remain incompletely understood. </p><p> Methods: The effect of M1 macrophages on the proliferation ability and cell viability of PDAC cells was evaluated by Cell Counting Kit-8 (CCK-8) cell proliferation assay, cell clone formation assay, and flow cytometry. Western blot, qRT-PCR, confocal microscope, RNA-sequencing, and transmission electron microscope were performed to assess lipid peroxidation and ferroptosis level of PDAC cells in the context of M1 macrophage or TNF-α.</p><p> Results: M1 macrophages inhibited cell proliferation and promoted cell death of PDAC cells, in which ferroptosis played a vital role. Mechanistically, Tumor Necrosis Factor-alpha (TNF-α) released by M1 macrophages binds to the TNFR1 receptor on pancreatic cancer cells, activating the p38 MAPK signaling, which upregulates Acyl-CoA Synthetase Long-chain family member 4 (ACSL4) expression, a critical lipid metabolism enzyme linked to ferroptosis, thereby promoting ferroptosis. Knockdown of ACSL4 or TNFR1 significantly reduced TNF-α-induced ferroptosis. Discussion: TNF-α is a major inflammatory cytokine and is mainly generated by macrophages and T lymphocytes. It is involved in many pathological processes, such as inflammatory diseases, autoimmune diseases, and cancer. Studies have shown that the administration of recombinant TNF-α can induce tumor regression in mice with sarcomas. In our study, systemic injection of TNF-α slowed the tumor growth in nude mice, but with no significant difference compared with the control group, which may partially be attributed to its angiogenic activity. TNF-α signals via two distinct membrane-binding receptors, TNFR1 and TNFR2, which regulate various diseases. In pancreatic cancer, the role of TNF-α is complex and poorly understood. In a previous study, Chopra et al. found that exogenous systemic administration of human TNF-α, which interacted with murine TNFR1, significantly increased overall tumor growth in the Panc02-PDAC model. Intriguingly, the loss of TNFR1 led to an impediment of immune cell infiltration into the tumor and impaired immunosurveillance, which accelerated tumor growth. This suggests that TNFR1 exerts both pro-tumoral and anti-tumoral functions in the Panc02-PDAC model, but the overall outcome is likely dependent on the spatiotemporal availability of TNF-α. However, systemic TNF-α injection can lead to severe side effects in animals, limiting its further application. In a recent study, TNFR2 was found to promote tumorigenesis and progression in the KPC-PDAC model. Knockdown of TNFR2 or pretreatment with an anti-TNFR2 antibody could significantly slow the tumor progression and incidence. In our study, TNFR2 was found to have a low expression in pancreatic cancer cells and was barely detected with the failure of knockdown. However, the cell lines used in the former study were established from a KPC mouse model, while our experiments were conducted using human PDAC cell lines. Contrary findings are possible as cell lines originate from two different species. However, we will further investigate the mechanism of this difference.</p><p> Conclusion: In summary, this study revealed that M1 macrophages could induce ferroptosis in pancreatic cancer cells through secreting TNF-α, indicating a potential therapeutic option for PDAC.</p>.
