Chemo-immunotherapy as a reshaper of immunosuppressive activity and gene expression of circulating monocytes in advanced non–small cell lung cancer.

Abstract

e14538 Background: Circulating monocytes (CM) may acquire immunosuppressive activity (IA) and contribute to resistance to immune checkpoint inhibitors (ICIs), while the ICIs impact on CM function is not fully clarified. We previously reported that ICIs can downregulate cellular FLICE-inhibitory protein (c-FLIP), a mediator of CM IA, in non-progressing non–small cell lung cancer (NSCLC) patients (pts). Methods: Pts with advanced NSCLC treated with first-line ICIs, alone (I) or with chemotherapy (CI), were prospectively enrolled at the University of Verona. Blood samples were collected at baseline (T0) and after 3 months (T1). Pts were classified as responders (R; PFS ≥6 months) or non-responders (NR; PFS < 6 months). CM were isolated and co-cultured with anti-CD3/CD28–activated healthy donor PBMCs to assess IA by flow cytometry. Group comparisons used unpaired t tests (p < 0.05). c-FLIP interactome was analysed by liquid chromatography–mass spectrometry in nuclear and cytoplasmic fractions of FLIP-overexpressing monocytes. RNA-seq libraries were performed with TruSeq RNA Prep Kit v2 and sequenced with NovaSeq 6000. Results: From 2021 to 2025, 80 pts were enrolled: 57 evaluable for CD3⁺ T-cell proliferation at T0, 40 receiving CI (70.2 %) and 17 receiving I (29.8%). Between CI-treated pts, 21 (52.5%) were classified as R and 19 (47.5%) as NR. CM from R pts showed higher IA than NR (p = 0.03). At T1, considering specifically pts receiving CI, CM from R pts showed reduced IA after treatment (p = 0.04), whereas IA increased in NR (p = 0.03). Based on RNA-seq analysis, transcriptional profile of CM from R is affected by CI (T1 vs T0, fdr (padj) < 0.1; up-regulated genes: 587, down-regulated genes: 476). Gene-set enrichment analysis revealed a strong modulation of lipid metabolism (MLXIPL, SPHK1, NR1D1) and biological processes related to cell-differentiation (FOSL2, NR4A1, MAFB). Analysis of nuclear and cytoplasmic c-FLIP interactome identified lactate dehydrogenase A (LDH-A) as a c-FLIP–interacting protein in both compartments. According to TCGA analysis, high LDH-A expression in the tumor microenvironment was associated with worse survival in lung adenocarcinoma (p < 0.0001). Accordingly, in our cohort of CI-treated patients, R had significantly lower baseline LDH levels than NR (p = 0.0071). Conclusions: CI reshapes circulating monocytes in NSCLC, reducing IA and modifying transcriptional profiles associated with CM differentiation in responders. Moreover, LDH levels could serve as an immunologically plausible and easy-to-use tool to select pts. Clinical trial information: 1839CESC.