Efficacies of Protein S, Pan-Notch Inhibitor CB-103, and Gemcitabine in Decreasing Cancer Cell Spheroid Growth

Introduction

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer with limited treatment options. Patients with PDAC experience a hypercoagulable state and 7.4% experience a form of venous thromboembolism (VTE) within 1 year of diagnosis, compared with other cancer patients who have a risk of 3.1%. The elevated risk of VTE in PDAC patients is likely associated with dysregulation of anticoagulation proteins, such as Protein S (PS). The Majumder laboratory showed that an exogenous increase in PS inhibited growth of PDAC cell lines PANC-1, BxPC-3, and MiaPACA-1. Furthermore, we have shown that high concentrations of free PS, particularly 150 nM, significantly decreased PANC-1 spheroid formation.

Notch, a key regulator of metabolism, displays aberrant signaling in several cancers, including pancreatic cancer. The pan-Notch inhibitor CB103 promotes apoptosis in pancreatic cancer.

In this study, we measured alone and in combination the efficacy of PS, CB103, and gemcitabine, the standard treatment for PDAC, in inhibiting PDAC spheroid formation.

Methods

Primary PANC-1 spheroids were induced from 3.7x10⁶ PANC-1 cells in 0.24% methylcellulose DMEM media on an ultra-low attachment 6-well plate. After incubation for 7 days, PANC-1 spheroids were harvested, and secondary spheroids were placed on a fresh ultra-low attachment 6-well plate with the following additions: 5% DMSO (vehicle control, no treatment),150 nM PS, 250 nM gemcitabine, 5 uM CB103. Combination treatments at the aforesaid concentrations were PS + gemcitabine, CB103 + PS, CB103 + gemcitabine, and all three combined. A representative number of spheroids from each well was washed with PBS and placed in triplicate in an ultra-low attachment 96-well plate. One day after, the cells were imaged, and cell count was determined with ImageJ.

Results

For all treatments, we measured a statistically significant (p <0.0001) reduction in spheroid formation. Individually, PS was the most effective in decreasing spheroid count, and the combination treatment of all three was the most effective overall in decreasing spheroid count.

Conclusions

We found that PS, a natural anticoagulant, had greater efficacy in decreasing PANC-1 spheroid growth compared with the efficacy of either CB103 or gemcitabine, the latter being the standard of care for patients with PDAC. Furthermore, combined treatments synergistically decreased PANC-1 spheroid growth. To proceed with clinical translation of these treatments, we need additional research to fully explain the activities of the three agents toward expression of cancer stem cell markers on PANC-1 and other PDAC cell line spheroids.

No relevant conflicts of interest to declare.