Chemotherapy-induced immunogenic hub restores NK cell activity via P2X7R in NSCLC

Background. Advanced non-small cell lung cancer (NSCLC) lacking actionable molecular alterations remains a major clinical challenge, with immunotherapy and chemoimmunotherapy representing the main treatment options. Although natural killer (NK) cells are key effectors of innate antitumor immunity, their role within the NSCLC tumor microenvironment (TME) remains incompletely understood. Methods. NK-92 cells and NSCLC A549 and H1299 lines were cultured in 2D, 3D, in co-cultures, and exposed to radiotherapy (5-7.5 Gy) or chemotherapy (gemcitabine, pemetrexed, cisplatin). Functional effects were evaluated by MTS, Annexin-V, β-galactosidase assays, synergism analyses, and confocal imaging. Gene expression was assessed using quantitative real-time PCR (qRT-PCR) and digital droplet PCR (ddPCR). Spatial transcriptomics via Nanostring Digital Spatial Profiling (DSP) was performed on tissue microarrays (TMAs) prepared from NSCLC patients tissues collected at IHU RespirERA (Nice, France), then bulk RNA sequencing was conducted. Results. We investigated whether conventional anticancer treatments and purinergic signaling could modulate NK cell-mediated antitumor activity. Chemotherapy, but not radiotherapy, significantly altered the expression of NK cell-activating ligands on NSCLC cells. Accordingly, co-culture experiments showed that pre-treatment with gemcitabine or pemetrexed enhanced NK cell-mediated tumor cell killing. These agents also increased tumor immunogenicity by upregulating NK-activating ligands and inducing a senescence-associated secretory phenotype. Digital spatial profiling of patient-derived NSCLC tissues revealed higher P2RX7 expression in tumors lacking actionable alterations, which was associated with NK cell-related transcriptional signatures. In vitro, P2RX7 expression decreased following co-culture of NK-92 cells with chemotherapy-pretreated NSCLC cells. Moreover, pharmacological inhibition of P2X7 receptor with AZ10606120 promoted NK-92 cell infiltration into NSCLC spheroids. Combination experiments further demonstrated that AZ10606120 synergized with pemetrexed or cisplatin to enhance NK cell-mediated cytotoxicity. Conclusions. Overall, our findings provide strong rationale for combinatorial strategies integrating chemotherapy with NK-targeted immunomodulation. These results further identify purinergic signaling as a promising therapeutic axis to expand treatment options for this patient population.