| Summary: | Background: Cancer-associated thrombosis (CAT) and venous thromboembolism (VTE) are frequent cancer-related complications associated with high mortality; thus, this urges the identification of predictive markers. Immune checkpoint inhibitors (ICIs) used in cancer immunotherapy allow T-cell activation against cancer cells. Retrospective studies showed increased VTE following ICI administration in some patients. Non-small cell lung cancer (NSCLC) patients are at high risk of thrombosis and thus, the adoption of immunotherapy, as a first-line treatment, seems to be associated with coagulation-fibrinolysis derangement. Methods: We pharmacologically modulated NSCLC cell lines in co-culture with CD8<sup>+</sup> T-cells (TCD8<sup>+</sup>) and myeloid-derived suppressor cells (MDSCs), isolated from healthy blood donors. The effects of ICIs Nivolumab and Ipilimumab on NSCLC cell death were assessed by annexin V and propidium iodide (PI) flow cytometry analysis. The potential procoagulant properties were analyzed by in vitro clotting assays and enzyme-linked immunosorbent assays (ELISAs). The metabolic remodeling induced by the ICIs was explored by <sup>1</sup>H nuclear magnetic resonance (NMR) spectroscopy. Results: Flow cytometry analysis showed that TCD8<sup>+</sup> and ICIs increase cell death in H292 and PC-9 cells but not in A549 cells. Conditioned media from NSCLC cells exposed to TCD8<sup>+</sup> and ICI induced in vitro platelet aggregation. In A549, Podoplanin (PDPN) levels increased with Nivolumab. In H292, ICIs increased PDPN levels in the absence of TCD8<sup>+</sup>. In PC-9, Ipilimumab decreased PDPN levels, this effect being rescued by TCD8<sup>+</sup>. MDSCs did not interfere with the effect of TCD8<sup>+</sup> in the production of TF or PDPN in any NSCLC cell lines. The exometabolome showed a metabolic remodeling in NSCLC cells upon exposure to TCD8<sup>+</sup> and ICIs. Conclusions: This study provides some insights into the interplay of immune cells, ICIs and cancer cells influencing the coagulation status. ICIs are important promoters of coagulation, benefiting from TCD8<sup>+</sup> mediation. The exometabolome analysis highlighted the relevance of acetate, pyruvate, glycine, glutamine, valine, leucine and isoleucine as biomarkers. Further investigation is needed to validate this finding in a cohort of NSCLC patients.
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