Interaction between NSCLC Cells, CD8⁺ T-Cells and Immune Checkpoint Inhibitors Potentiates Coagulation and Promotes Metabolic Remodeling—New Cues on CAT-VTE

Abstract

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+ T-cells (TCD8+) 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 1H nuclear magnetic resonance (NMR) spectroscopy. Results: Flow cytometry analysis showed that TCD8+ and ICIs increase cell death in H292 and PC-9 cells but not in A549 cells. Conditioned media from NSCLC cells exposed to TCD8+ 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+. In PC-9, Ipilimumab decreased PDPN levels, this effect being rescued by TCD8+. MDSCs did not interfere with the effect of TCD8+ 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+ 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+ 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.

Keywords

• cancer-associated thrombosis (CAT)
• venous thromboembolism (VTE)
• immune checkpoint inhibitors (ICIs)
• non-small cell lung cancer (NSCLC)
• metabolic remodeling
• tissue factor (TF)