01.13 Arteriolar Differentiation of Vascular Endothelial Cells in Malignant Progression of PNETs

A. Akil1, V. G. Bolus1, A. W. Beck1, H. Chen1, B. Ren1 1The University of Alabama at Birmingham,Surgery,Birmingham, ALABAMA, USA

Introduction: Pancreatic neuroendocrine tumors (PNETs) are highly vascularized and heterogeneous neoplasms, which are characterized by high levels of VEGF and its receptors, the potential driver in PNET metastasis. An antiangiogenic drug known as sunitinib has been approved for the treatment of PNETs. However, the therapeutic efficacy is limited because of relapse and metastasis. Therefore, tailoring antiangiogenic therapy to patients requires novel insight of tumor angiogenesis. Our previous studies suggested that PKD-1 signaling stimulates arteriolar differentiation (a key process in functional angiogenesis) via the Notch pathway and promotes tumor progression. We hypothesize that PKD-1-stimuated arterial differentiation plays an essential role in PNET progression by development of a unique vascular niche.

Methods: To test this hypothesis, we developed a co-culture system for growing human microvascular endothelial cells (HMVECs) with PNET cells using a Boyden Chamber technique. To define the molecular mechanisms by which PKD-1 signaling regulates PNET progression, we knocked down endogenous PKD-1 expression and characterized malignant behavior of PNET cells using Clonogenic and tumor invasion assays. Furthermore, real-time RT-qPCR was used to evaluate the transcriptional expression of genes associated with arteriolar differentiation and tumor progression.

Results:Compared with the scramble control, PKD-1 knockdown in HMVECs decreased the formation of colonies and impaired the invasive potential of PNET cells. Unexpectedly, co-culture of control HMVECs with PNET cells showed clear formation of many colonies with tumorsphere-like morphology. However, when these cells co-cultured with PKD-1-deficient HMVECs, the number of spheres was significantly reduced.  At molecular levels, knocking down PKD-1 in HMVECs reduced mRNA expression of ALDH1A1, VEGFR2 and Notch1 but increased CD36 expression when co-cultured with PNET cells. Intriguingly, silencing PKD-1 in HMVECs downregulated mRNA levels of KLF2, Myc, ALDH1 and Snail 1 (a transcriptional factor for epithelial mesenchymal transition) in PNET cells. 

Conclusion:This study indicates that there is a crosstalk between endothelial cells and PNET cells to promote arteriolar differentiation, in which PKD-1 signaling may play a key role. Cancer cell-mediated arteriolar differentiation via the PKD-1 pathway may contribute to the progression of PNETs by serving as a unique vascular niche in the tumor microenvironment.