Cancer Associated Thrombosis Alters Neutrophils to Promote Pancreatic Ductal Adenocarcinoma Progression

Introduction: The occurrence of cancer-associated thrombosis (CAT) is associated with poor prognosis and increased mortality from the cancer itself suggesting that venous thromboembolism (VTE) is more than just an unfortunate side effect of cancer. While it is well recognized that cancer is associated with an increased risk for venous thrombosis, it is still not understood how CAT may alter subsequent cancer growth and progression. Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a <5% 5-year survival rate and a high risk of CAT. Given the role of the innate immune system in CAT and tumor growth, we posited that the cells of the innate immune system, specifically neutrophils are altered by VTE to facilitate tumor growth.

Materials and Methods: To investigate the role of cancer associated thrombosis (CAT) in PDAC progression, we developed a unique mouse model of PDAC and CAT. C57BL/6J mice were injected intra-pancreatically with PANO2 cells, a murine pancreatic cancer cell line with luciferase activity. VTE was induced one week later with complete inferior vena cava ligation. Tumor growth was monitored by detecting PANO2 bioluminescence. Neutropenia was generated with Ly6G antibody mediated neutrophil depletion strategy for two weeks. Neutrophil transcriptome was assessed via RNA sequencing and the immune cells in the tumor microenvironment (TME) were characterized through flow cytometry and immunohistochemistry. T cell apoptosis assays were performed by co-culturing CD8⁺T cells (derived from the spleen) with bone marrow or tumor associated neutrophils.

Results:

1. C57BL/6J mice with PDAC and CAT (by IVC ligation) developed significantly larger tumors as compared to mice with only PDAC demonstrating that CAT enhances tumor growth.

2. Neutropenia (Ly6G injection) significantly decreased the tumor growth in PDAC + CAT mice as compared to similar non-Neutropenic mice (control IgG injection). Interestingly, in the PDAC only mice, neutropenia resulted in increased tumor growth as compared to their control non-neutropenia counterparts. These results indicate that while neutrophils have an anti-tumor effect in PDAC, the presence of a thrombus changes the activity of these cells so that now neutrophils contribute to increased tumor growth in the presence of CAT.

3. RNA-seq analysis of peripheral blood neutrophils from PDAC + CAT mice showed that these neutrophils exhibited altered gene expression signatures specifically affecting differentiation and inflammatory pathways as compared to PDAC only, VTE only or control mice.

4. No difference in total neutrophil or macrophage numbers, but decreased CD8+ T cells were noted in the TME of PDAC + CAT as compared to PDAC only mice.

5. Co-culturing CD8⁺T cells with TANs isolated from same mice showed an increase in the apoptosis of T cells in the presence of TANs from PDAC + CAT as compared to PDAC only mice signifying that CAT facilitates neutrophils with an enhanced capacity to induce CD8⁺ T cell apoptosis.

Conclusion: Our results are the first to demonstrate that CAT is associated with altered neutrophil activity that affects the TME and facilitates tumor growth. These results suggest that neutrophils may serve as a novel targetable pathway to improve care for pancreatic cancer patients with CAT. Ongoing studies are exploring the molecular mechanisms involved in CAT-directed neutrophil alteration and neutrophil-mediated T cell apoptosis.

No relevant conflicts of interest to declare.