Regenerating islet derived Family Member 4 (REG4) does not influence parameters of hemostasis in colorectal cancer.

BACKGROUND

Colorectal cancer (CRC) patients are at increased risk of developing venous thromboembolism (VTE), leading to high morbidity and mortality. The exact mechanism remains unknown, which complicates risk prediction and treatment. Previously, using RNA-sequencing, Regenerating islet derived Family Member 4 (REG4) was identified to be upregulated in tumor cells from CRC patients with VTE compared to tumor cells from CRC patients without VTE.

OBJECTIVE

Here we aim to functionally validate the role of REG4 in cancer-associated thrombosis (CAT) using different in vitro tests of thrombosis and hemostasis.

METHODS

Recombinant purified protein (rREG4), as well as REG4 overexpressing human colorectal cell lines were used to characterize the possible role of REG4. Prothrombin time (pT), activated partial thromboplastin time (aPTT) in normal pooled plasma (NPP) and whole blood and thrombin generation in NPP were determined in the presence of rREG4. In addition, the effect of rREG4 and RKO-REG4 expressing cells on plasma clot turbidity after exposure to human umbilical vein endothelial cell (HUVEC) was assessed by a turbidimetric assay, qPCR and Electric Cell-substrate Impedance Sensing (ECIS). In an organ-on-a-chip-model for cancer-associated hypercoagulability, RKO cells were co-cultured with vessel-forming HUVECs, after which thrombin generation was assessed.

RESULTS

rREG4 did not alter pT and aPTT clotting times nor the thrombin generation potential in NPP and whole blood. Conditioned medium of RKO-REG4 secreting cells did not alter the thrombin generation potential, compared to the negative control (mock transfected RKO cells). In addition, no effect was observed on in vitro turbidimetric clot formation or on ECIS after endothelial cell stimulation. In line with this, using a cancer-associated hypercoagulability-on-a-chip model, RKO-REG4 cells did not further influence endothelial barrier integrity, nor thrombin generation potential, compared to control RKO cells.

CONCLUSIONS

Extensive in vitro and microfluidic methods showed no effect of REG4 on coagulation parameters in CRC. Further research is needed into the mechanism behind CAT, as its understanding will help in better risk prediction and treatment.