Abstract C014: Degradation of extracellular trap DNA sustains anti-tumor immune responses in breast cancer

Abstract

Abstract Increased extracellular DNA (exDNA) levels in the blood are a hallmark of metastatic cancer, arising from tumor lysis, apoptosis, necrosis, and neutrophil extracellular traps (NETs) released by tumor-expanded neutrophils. NETs, web-like chromatin structures extruded by neutrophils to trap pathogens, are highly immunogenic due to their DNA and histone content and their persistent interaction with dendritic cells. Tumor cells can also release extracellular traps. In myeloid leukemia, blasts release nuclear content via traps to activate coagulation or sustain myeloproliferation. Extracellular traps have been found in NPM1 mutant AML patients, with mutant NPM co-localizing with histones along exDNA traps. Forcing trap extrusion in NPM mutant leukemia cells has been used to create dendritic cell-based vaccines that break tolerance against NPMc antigens. This suggests that NETs in the tumor microenvironment (TME) may promote tolerance, with breaking tolerance requiring consistent NET formation and dendritic cell interaction. NETs are also seen in solid tumors, such as triple-negative breast cancer (TNBC), where they aid in cancer cell migration, invasion, and awakening of circulating tumor cells. However, the capacity of tumor cells to directly extrude DNA traps is not fully understood. To investigate this, various human and murine breast cancer cell lines were seeded on poly-D-lysine coated slides, stained with DAPI and Sytoxgreen, and observed via confocal microscopy. Tumor cells extruded DNA traps within 4 hours without stimuli. Notably, trap extrusion correlated with cell line aggressiveness and was abolished by DNase, indicating dsDNA composition. The absence of traps in 24-hour cultures suggests DNA extrusion may help rare tumor cells survive and proliferate. In line, DNase treatment reduced tumor proliferation and increased apoptosis. To assess the significance of DNA traps in vivo, BALB/c mice were injected with the highly metastatic 4T1 clone 5 breast cancer model, with or without DNase treatment, and monitored starting 5 days post-injection and weekly until day 28. Histological analysis at the injection site 5 days post-injection showed that DNase treatment reduced both local trap formation and tumor cell proliferation, as indicated by BrdU incorporation. Moreover, immunohistochemistry revealed that in DNase-treated mice, initial tumor growth was followed by complete tumor regression, accompanied by local and systemic CD8 T cell activation. Correspondingly, although a few metastases were detectable at day 14 in DNase-treated mice, no metastases were observed by day 28. Although it remains unclear whether tumor-derived or immune cell-derived traps are most relevant in our model, the results demonstrate that their presence in the TME promotes tolerance despite the inherent immune adjuvant properties of the traps. This suggests that DNase treatment could be particularly effective in combating early tumor development or relapse when used as part of a combination therapy designed to sustain anti-tumor immune responses. Citation Format: Sabina Sangaletti, Paola Portararo, Laura Botti, Valeria Cancila, Claudio Tripodo, Mario P. Colombo, Claudia Chiodoni. Degradation of extracellular trap DNA sustains anti-tumor immune responses in breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C014.