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Abstract
Background Primary human organoids have been shown to be predictive for selectivechemotherapy and thus be a valuable model for cancer research. One of manytumor evasion strategies in response to DNA-damaging chemotherapy treatment isthe induction of senescence to acquire a state of resistance. Fortunately,natural killer (NK) cells recognize senescent tumor cells, get activated andtrigger tumor killing in assistance of T-cells, as it was shown in mice studiesand cell lines. Additionally, it has been described that activation of theSTING pathway strongly enhance NK-cell responses. Therefore, we hypothesized,that STING-activated NK-cells are superior in killing chemotherapy-inducedsenescent tumor organoids. To test this hypothesis in a primary humandisease-relevant model, we established a complex system consisting of tumor organoids,matched primary cancer-associated fibroblasts (CAFs) and immune cells.
Materials and Methods To establish complex organoid cultures, we investigated various air-liquid-interface(ALI) culture conditions of primary tumour spheroids, CAFs and peripheral bloodmononuclear cells (PBMCs). Senescence was induced by Etoposide treatment and wasverified by β-galactosidase staining. Immune cells were activated by either ionomycinand PMA, or a STING agonist ADU-S100.
Results Immune cell viability was preserved for 48 hours in the established ALI cultureconsisting of PBMCs and organoids in one phase and fibroblasts in a secondcompartment. Functional applicability of the system was evaluated through ionomycinand PMA induced immune cell activation which resulted in tumor cell death asquantified by zombie violet positive staining. Confocal microscopy further verifiedimmune cell infiltration and immune cell mediated disintegration of organoids.Senescence was inducible by DNA-damaging chemotherapy in 3Dco-culture of tumor organoids and CAFs. Moreover, a combination of senescenceinduction by DNA-damaging chemotherapy and subsequent STING-pathway activation ledto a more pronounced NK-cell activation (CD69) and degranulation (CD107a) incontrast to non-senescent controls.
Conclusions We developed a complex 3D culturesystem of tumor, stromal and immune cells to mimic the tumor microenvironment andto assess the impact of senescent organoids on STING-activated immune cells in a primary human model.
D. Ammon: None. M. Fabits: None. M. Sykora: None. J. Kabiljo: None. A. Theophil: None. V. Atanasova: None. H. Dolznig: None. M. Bergmann: None.
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