Necroptosis triggers inflammatory interferon signatures in patient-derived metastatic breast cancer organoids.

Breast cancer (BC) is the most common type of cancer among women worldwide and underlies relapse, disease progression, and metastasis. Resistance to chemotherapy and programmed cell death (PCD), including apoptosis, strongly affects BC therapy success and remains a major challenge. Although necroptosis, a lytic, and via damage-associated molecular patterns (DAMP) release, highly immunogenic mode of PCD, might overcome apoptosis resistance, there is an urgent need for physiological and translational human models to model necroptosis and PCD resistance in BC. Here, we apply 3D patient-derived, metastatic human mammary organoids (hMOs) to model apoptosis resistance, necroptosis, and inflammatory signaling using single-cell CITE-sequencing, time-lapse live cell brightfield, and immunofluorescent confocal microscopy, as well as biochemical approaches. Smac mimetic-triggered apoptosis could be confirmed in a panel of BC hMOs. Upon inducing apoptosis resistance with caspase inhibition, BC hMOs rapidly undergo necroptosis with profound MLKL phosphorylation. Necroptotic cell death was preceded by prominent transcriptional upregulation of inflammatory cyto- and chemokines, including interferons, that activate natural killer cells. Finally, necroptosis and the expression and release of inflammatory messengers in metastatic BC hMOs could be attenuated upon the inhibition of linear ubiquitination. We describe a novel experimental platform to model PCD, inflammation, and necroptosis that allows therapeutic screening to overcome chemotherapy resistance in patient-derived metastatic BC hMOs. With this platform, we identified necroptosis-induced interferon signaling, suggesting Smac mimetics and necroptosis as a potential immunotherapy strategy against metastatic BC.