Study of the immune synapse generated by Cytokine-induced memory-like (CIML) NK cells targeting clustered NSCLC cells and Cancer Stem Cells (CSCs)

Lung cancer, the second most prevalent malignancy worldwide, remains highly lethal due to late diagnosis and a significant propensity to metastasize. Circulating tumor cell (CTC) clusters play a critical role in the process of metastasis generation by transporting cancer stem cells (CSCs) and shielding them from immune surveillance. Among innate immune effectors, Natural Killer (NK) cells represent a promising tool in cancer immunotherapy due to their ability to recognize and eliminate tumor cells through formation of a lytic immunological synapse (IS). However, some isolated tumor cells reduce IS efficacy by remodeling the actin cytoskeleton in the cell-to-cell contact. It is presently unknown whether this process, termed actin response (AR), could also be induced in organized cell aggregates, and could be modulated by different immune effector cells. We have generated a model of Non-Small-Cell Lung Cancer (NSCLC) cell spheroids, mimicking CTC clusters, and studied their interactions with NK cells. By setting up co-culture experiments, we comparatively analyzed NK cells classically stimulated with IL2 or with IL15+IL12+IL18 (Cytokine-Induced Memory-Like - CIML-NK, newly tested in clinics). We first demonstrated that AR occurs and is favored in tumor cell clusters. Next, analyzing effector-tumor cell conjugates by different imaging techniques, we found that CIML-NK cells present an advantage over IL2-NK cells in limiting AR of spheroid-derived cells and, specifically, of their CSC component. Overall, our data have important clinical implications: (i) specific effector cell types/preparations can differently modulate AR, with CIML-NK cells showing increased efficacy in blocking such escape mechanism, especially towards CSCs; (ii) the demonstration of AR in organized tumor cell clusters indicates the regulation of such process as a new field of study for the identification of effective therapeutic targets.