Systemic inhibition of Dll4 results in a vast reduction of cancer metastasis. Whether this effect is endothelial-mediated remains to be clarified. Therefore, we proposed to analyze the impact of endothelial Dll4 loss-of-function on metastasis induction on three early steps of the metastatic process, regulation of epithelial-to-mesenchymal transition (EMT), cancer stem cell (CSC) frequency and circulating tumour cell (CTC) number.
Lewis Lung Carcinoma (LLC) cells or LLC- green fluorescent protein marked were used to model mouse tumour metastasis in vivo, by subcutaneous transplantation into endothelial-specific Dll4 loss-of-function mice.
We observed that endothelial-specific Dll4 loss-of-function is responsible for the tumour vascular regression that leads to the reduction of tumour burden. It induces an increase in tumoural blood vessel density, but the neovessels are poorly perfused, with increased leakage and reduced perivascular maturation. Unexpectedly, although hypoxia was increased in the tumour, the number and burden of macro-metastases was significantly reduced. This is likely to be a consequence of the observed reduction in both EMT and CSC numbers caused by the endothelial-specific Dll4 loss-of-function. This multifactorial context may explain the concomitantly observed two-fold reduction of the circulating tumour cell count. Furthermore, our gene transcription analysis suggests that endothelial Dll4/Notch function mediates tumour hypoxia-driven increase of EMT. We observed that the downregulation of Dll4/Notch1/Hey1 signalling caused a reduction in Snail-1 and Twist expression, known inducers of EMT.
These findings show that the effect of Dll4 inhibition in suppressing cancer metastasis is, at least, partially endothelial-mediated. This is accomplished through the arrest of the EMT process and reduction of CSC clone selection in the primary tumour, regulated by non-cell-autonomous endothelial signalling. Therefore, endothelial Dll4 may constitute a promising target for the prevention of metastasis.
Clinical trial identification
Legal entity responsible for the study
CIISA - Centro de Investigação Interdisciplinar em Sanidade Animal.
This work was supported by the Portuguese Foundation for Science and Technology (FCT), grants PTDC/CVT/115703/2009 to AD; PTDC/SAU-ONC/116164/2009 and PTDC/SAU-ONC/121742/2010 to AT. CIISA has provided support through project UID/CVT/00276/2019, funded by FCT. LM is a PhD student supported by a studentship from FCT (SFRH/BD/74229/2010).
All authors have declared no conflicts of interest.