In this study we demonstrate that Notch4 expression is upregulated in the vasculature of mammary tumors. Furthermore, our results suggest that host Notch4 plays a role in the early emergence of MMTV-PyMT breast tumors following transplantation. We also found that host Notch4 is required for initial tumor vascular perfusion, but not vessel sprouting. To our knowledge, this is the first report demonstrating the functional relevance of host Notch4 upregulation in tumorigenesis and tumor vessel perfusion. Our findings shed light on the role of Notch4, independent of other Notch receptors, in tumor-host interactions.
Our results show increased levels of Notch4 in the blood vessels of mouse and human breast tumor tissues. Expression of Notch receptors and ligands has been described in a wide variety of different tumor types, such as colorectal, prostate, liver, pancreatic and breast cancer [23, 24] and a role for Notch4 in regulating breast cancer stem cell activity has been proposed . Mittal et al. reported that the levels of Notch receptors (1, 2, and 4) and ligands (Jagged 1, 2, Dll1, and 4) are increased in human breast cancer compared to normal breast tissue . Recently, a study by Speiser and collaborators showed increased Notch1 and Notch4 levels in tumor epithelial cells and vascular endothelial cells in triple-negative breast cancer samples , therefore highlighting the relevance of Notch4 expression in the vasculature. However, in our studies, we did not observe Notch4 staining in either human (Figure 1c) or murine tumor cells (Figure 1a, ix, xii and 1b, iii), but rather observed Notch4 upregulation in the tumor vasculature. We used a well-characterized Notch4-specific antibody  and verified its specificity using Notch4 knockout tissues (Additional file 1: Figure S1). Our study demonstrates Notch4 upregulation in the vasculature of both mouse models of mammary adenocarcinoma and human breast cancer. It is possible that different types and grades of breast carcinoma present different Notch4 expression levels and distribution. However, in our study, our mouse model provides evidence that host, likely vascular, Notch4 plays a role in breast cancer development. Consistent with our finding that Notch4 is expressed in the tumor vasculature, the Notch ligand Dll4 is detected in the vessels of infiltrating human breast adenocarcinoma samples , making it a possible ligand for Notch4 in tumor vasculature.
Orthotopic transplantation of mammary tumor cells is a well-established model for in vivo studies of breast tumorigenesis, and we chose this approach because it allowed us to study host Notch4-mediated effects on tumorigenesis independent of Notch4 activity in the tumor cell compartment. We demonstrate a contribution of the tumor microenvironment, namely the host Notch4, to tumorigenesis. We also pinpoint that the tumorigenic defect lies at the tumor onset following transplantation. It is well documented that the growth of solid tumors depends on the development of new vasculature . Inhibition of members of the Notch signaling pathway leads to an increase in “non-productive” angiogenesis, characterized by a reduction in vessel perfusion despite an increase in vessel sprouting [9–11, 31]. We thus examined vascular perfusion in tumors grown in Notch4
hosts. Since both the mammary gland and its vasculature appear normal in Notch4
mice, we reasoned that any vascular defects in tumors from Notch4
hosts must be due to abnormalities that occur during tumorigenesis, and not as a result of preexisting vascular defects. We observed that vessel perfusion was reduced in tumors grown in Notch4
vs. wild type hosts. This observation is consistent with many reports of Notch pathway inhibition leading to reduced perfusion [9–11, 31].
Although poor tumor vessel perfusion correlated significantly with delayed tumor onset at early time points after transplantation, our results alone do not provide causal proof that reduced vessel perfusion leads to delayed tumor onset in Notch4−/− mice. Given the complex dynamics in tumor-host interaction and tumor microenvironment, we cannot rule out the possibility that vascular Notch4 (and other Notch pathway proteins) may regulate tumor onset by mechanisms that are independent of vessel perfusion. It is also possible that differences in transplantation-associated immune responses contribute to the delayed tumor onset in Notch4
hosts. Both tumor-associated fibroblasts and tumor-infiltrating leukocytes have been shown to play an important role in tumor onset and growth . Notch4 expression has been detected in immune cells of myeloid lineage . Although we detected Notch4 overexpression predominantly in the tumor vasculature, we cannot rule out the possibility that host myeloid cells may contribute to the difference in tumor onset between the two host genotypes.
Host Notch4 deficiency delays tumor onset and decreases initial perfusion, however, the growth of established tumors can ultimately progress in the absence of host Notch4. This result suggests that host Notch4 plays a unique role in the initiation of tumor onset after transplantation.
Surprisingly, our results indicate that Notch4 is dispensable for vessel sprouting in the tumor. Sprouting angiogenesis is a hallmark of tumor neovascularization, and Dll4/Notch1 signaling functions to inhibit vessel sprouting . Moreover, given that Notch1 appears to be the primary Notch receptor responsible for developmental angiogenesis , together with the results obtained using specific anti-Notch1 antibodies in tumors , Notch1 seems to be the predominant mediator of Notch signaling in tumor angiogenesis. It is therefore likely that the increased vascular network observed when inhibiting pan-Notch signaling or Notch ligands is mainly due to the inhibition of Notch1. Alternatively, it is possible that a subtle defect in vessel sprouting exists in the Notch4−/− tumor vasculature, but current methodologies are not sensitive enough to detect such subtle phenotype.