Ovarian cancer ascites impacts natural killer and innate lymphoid cell phenotype and function within the tumor microenvironment

Background Ovarian cancer (OC) is the most lethal gynecologic malignancy,
with disease progression critically shaped by the tumor microenvironment.
Ascitic fluid constitutes a complex cellular and soluble niche that
promotes tumor growth and immune evasion; however, the innate lymphoid
landscape within OC ascites remains poorly characterized. Methods We
performed single-cell RNA sequencing of natural killer (NK) cells isolated
from OC ascites to resolve innate immune heterogeneity and differentiation
states. Functional assays assessed NK cell cytotoxicity, degranulation,
and receptor expression following exposure to ascites, with or without
transforming growth factor-β (TGF-β) blockade. Proteomic profiling was
used to define the ascites cytokine milieu. Results We identified eight
distinct NK/innate lymphoid subclusters spanning cytotoxic, precursor,
early-like, tolerant/immunoregulatory, regulatory, proinflammatory, and
innate lymphoid cell (ILC) states. OC ascites were characterized by
depletion of cytotoxic and precursor NK cells and enrichment of
early-like, tolerant, regulatory, and proinflammatory populations.
Pseudotime trajectory analysis revealed impaired maturation toward
terminally differentiated cytotoxic NK cells. Notably, a functionally
exhausted innate lymphoid cell 2 (ILC2) population accumulated in ascites
and was associated with poor progression-free survival. Exposure of
healthy donor NK cells to ascites impaired degranulation and killing of
OVCAR-4 cells, reduced activating receptor expression (NKp30, DNAM-1),
increased inhibitory receptor expression (PD-1, LAG-3, TIGIT), and induced
a shift toward CD56highCD16low NK cells. Proteomic profiling revealed an
immunosuppressive, type-2–polarized cytokine milieu enriched in TGF-β and
ILC2-promoting mediators. Pharmacological TGF-β blockade partially
restored NKp30/DNAM-1–dependent NK cell cytotoxicity. Conclusions OC
ascites establishes a type-2–skewed immunoregulatory niche that
coordinately drives NK cell dysfunction and ILC2 accumulation. These
findings provide a systems-level framework linking ascites-derived cues to
innate immune remodeling and identify actionable pathways for restoring
antitumor immunity in ovarian cancer.