Cancer cells induce angiogenesis for themselves to obtain oxygen and nutrients for the growth and metastasis of solid tumors. Several anti-angiogenic agents have been approved for the treatment of various tumors to starve tumors to death by inhibiting the generation of new blood vessels in tumors [1]. However, it has been reported that the clinical benefits were limited due to the resistance to anti-angiogenic therapy when a selective anti-angiogenic agent was used as monotherapy [[2], [3], [4], [5]]. However, when an anti-angiogenic agent was used in combination with chemotherapeutics, the therapeutic benefits were improved [6,7].

To explain why the combination therapy showed positive results, Jain proposed the concept of vascular normalization using anti-angiogenic agents [8]. The blood vessels within tumor tissues are tortuous and leaky, and have irregular diameters and abnormal branching patterns [9,10]. The vascular endothelial cells are disorganized and loosely connected, with an incomplete basement membrane and an abnormal pericyte coat [11]. Thus, the tumor vasculature is structurally and functionally abnormal and incomplete, making it difficult to efficiently deliver drugs to tumor tissues [9,10]. In addition to the abnormal characteristics of tumor blood vessels, the tumor microenvironment, such as interstitial hypertension caused by heterogeneous blood flow, lack of lymphatic vessels, and abundant extracellular matrix (ECM) in tumor tissues, further prevents drug delivery to tumor cells [12]. Specifically, in breast cancer, the reduction of collagen, an ECM component, enhances the efficacy of nanotherapeutics [13,14]. The vascular normalization strategy using anti-angiogenic agents could improve these vascular abnormalities and subsequently the microenvironment in tumor tissues within a normalization window developed in an appropriate time- and dose-dependent manner. Then, the improved tumor microenvironment could allow drugs to be delivered to tumor cells and enhance their therapeutic efficacy [8,[15], [16], [17], [18]].

SU5416, a selective vascular endothelial growth factor (VEGF) receptor-2 kinase inhibitor, is a potent agent for the vascular normalization strategy. Due to its aqueous insolubility, SU5416 dissolved in DMSO and/or Cremophor EL is frequently administered into the vein or the peritoneal cavity at high doses [[19], [20], [21]]. Then, we have previously formulated the polyethylene glycol (PEG)-modified O/W emulsion loaded with SU5416 (PE-SU5416) and the single dose of PE-SU5416 successfully improved tumor vessels structurally and functionally and increased the distribution of subsequently administered PEG-modified liposomal paclitaxel (PL-PTX) to the core region of Colon-26 (C26) tumors, resulting in enhanced anti-tumor activity [22]. Considering that multiple injections are usually required for other agents [[23], [24], [25]], PE-SU5416 would be a promising formulation. However, the detailed mechanisms underlying successful outcomes of the vascular normalization strategy remain unclear, and the efficacy of selective anti-angiogenic agents would be dependent on the tumor subtype [26].

Triple-negative breast cancer is an aggressive type of breast cancer; however, there are no effective targeted therapies because of the lack of expression of human epidermal growth factor receptor 2, progesterone receptor, and estrogen receptor. Therefore, novel therapeutic strategies are required. In this study, we evaluated the therapeutic efficacy of vascular normalization strategy for 4T1, a triple-negative breast cancer, using PE-SU5416 and PL-PTX in 4T1 tumor-bearing mice, and successfully improved the anti-tumor activity of PL-PTX. To investigate the mechanisms underlying this improved therapeutic effect, we evaluated the effects of PE-SU5416 on the structure and function of tumor vessels. Furthermore, we evaluated cancer-associated fibroblasts (CAFs), a large population of the stromal cells within tumor tissues, because CAFs produce abundant ECM, which elevates interstitial fluid pressure (IFP) and prevents drug diffusion in tumor tissues [27,28]. Furthermore, it is also known that CAFs promote tumor growth and progression [[29], [30], [31], [32]] and are associated with poor prognosis in the lung [33] and breast cancers [34,35]. We successfully identified the possible involvement of CAFs and ECM in the improved anti-tumor effects of PL-PTX after PE-SU5416 treatment.