Dietary Tryptophan Contributes to Indoleamine 2,3-Dioxygenase 1(IDO-1)-Mediated Cancer-Associated Venous Thrombogenicity in Mice

Introduction: Cardiovascular disease (CVDs) is the second most common cause of death in cancer survivors, with venous thromboembolism (VTE) being a potentially fatal complication. Gastrointestinal cancer patients have the highest risk of cancer-associated VTE (CA-VTE). A high-protein diet, providing more tryptophan (Trp), is often prescribed to combat cancer-associated cachexia, and data indicate that Trp consumption in the United States is higher than recommended. Dietary Trp is converted to kynurenine (Kyn) through Indolamine-2,3-dioxygenase (IDO-1). Despite the clinical implications of CA-VTE, the influence of dietary protein and Trp on CA-VTE remains unclear. Here, we investigated the risk of VTE by dietary protein and Trp in a mouse model of cancer-associated thrombosis.

Methods: A syngeneic immunocompetent mouse model was generated using the mouse colon cancer cell line (MC38) in groups of 8-12-week-old male and female C57BL/6J mice. After reaching 400 mm³ xenograft size, mice were randomized into experimental groups receiving normal (20 kCal%) and high protein (40 kCal%) as well as normal (0.2%), zero (0%), and high Trp (1.2%) diets for 5 days. On day 3, inferior vena cava (IVC) ligation (stasis model) was performed. After 48 hours, clot weights were measured and normalized to body weights. The IVCs were analyzed for expression of pro- and anti-thrombotic mediators by Immunofluorescence and immunohistochemistry. LC/MS-based targeted metabolomics measured changes in Trp metabolites in serum across different experimental groups, both with and without cancer and IVC ligation.

Results: Normalized clot weights increased by ~40% in xenograft mice on a high protein diet (*P=0.0164) compared to normal diet. IVCs of xenograft mice on a high protein diet showed a 2-fold increase in tissue factor (TF) (P=0.0361) and a 1.5-fold increase in plasminogen activator inhibitor (PAI-1) (P=0.0255) compared to xenograft mice on a normal diet.

Xenograft-bearing mice on a high Trp diet had a ~30% increase in clot weights compared to normal Trp diet (P=0.001). No significant differences were observed in non-xenograft mice on different Trp diets. TF levels were higher in xenograft mice on a high Trp diet compared to normal Trp (P=0.0585) and zero Trp (P=0.0002) diets. Similarly, PAI-1 expression was higher in the high Trp diet group than in the normal and zero Trp groups (P<0.0001 and P<0.0001, respectively). Thrombomodulin (TM) expression was ~3-fold higher in the normal Trp diet group (P=0.001) and ~3.8-fold lower in the high Trp diet group (P<0.001) compared to the zero Trp diet group. Mice on normal and low Trp diets had significantly lower von Willebrand factor (vWF) levels compared to high Trp diet mice (P<0.001). Diet consumption did not differ between groups.

Xenograft mice on a high Trp diet had lower serum Trp levels (P=0.0508) but ~3-4-fold higher Kyn levels compared to normal or low Trp diets (P=0.0306, P=0.0269). Kyn levels were ~5-fold higher in xenograft-bearing mice on a high Trp diet compared to non-bearing mice. IDO-1 expression and activity were higher in xenograft-bearing mice (P=0.0037) and positively correlated with normalized clot weights (R²=0.324, P=0.007). High Trp diet in xenograft-bearing mice increased IDO-1 expression in endothelial cells compared to low (P<0.001) and normal Trp diets (P<0.0001). INCB23460, a potent IDO-1 specific inhibitor, reduced normalized clot weights by ~25-32% in normal Trp diet mice (P=0.0133) and by ~70% in high Trp diet mice (P=0.0011). INCB23460 also reduced Kyn levels in normal and high Trp diet mice (P=0.0199 and P=0.0501, respectively) and TF (P=0.004) and PAI-1 (P=0.03) expression compared to vehicle-treated groups.

Conclusion: Our results reveal a significant prothrombotic effect associated with a protein- or tryptophan-rich diet in a syngeneic colon cancer xenograft model. Specifically, a tryptophan-rich diet exacerbates CA-VTE, an effect markedly attenuated by an IDO-1 inhibitor. These findings suggest dietary interventions and IDO-1 targeted therapies could be crucial in managing thrombotic risks in cancer patients. Further research is warranted to explore these implications and refine therapeutic strategies.