Tumor vessel normalization by the PI3K inhibitor HS-173 enhances drug delivery

Highlights

• Tumor vessels are leaky and immature, which causes poor oxygen and nutrient supply, resulting in cancer cell metastasis.

• HS-173 caused vessel normalization in tumors, inhibiting vascular leakage and neoangiogenesis.

• In combination therapy, HS-173 increased blood perfusion and led to delayed tumor growth.

• Vessel normalization by HS-173 could be a novel therapeutic strategy for inhibiting tumor growth by improving drug delivery.

Abstract

Tumor vessels are leaky and immature, which causes poor oxygen and nutrient supply to tumor vessels and results in cancer cell metastasis to distant organs. This instability of tumor blood vessels also makes it difficult for anticancer drugs to penetrate and reach tumors. Numerous tumor vessel normalization approaches have been investigated for improving drug delivery into tumors. In this study, we investigated whether phosphoinositide 3-kinase (PI3K) inhibitors are able to improve vascular structure and function over the prolonged period necessary to achieve effective vessel normalization. The PI3K inhibitors, HS-173 and BEZ235 potently suppressed tumor growth and hypoxia, and increased tumor apoptosis in animal models. PI3K inhibitors also induced a regular, flat monolayer of endothelial cells (ECs) in vessels, improving stability of vessel structure, and normalized tumor vessels by increasing vascular maturity, pericyte coverage, basement membrane thickness, and tight-junctions. These effects resulted in a decrease in tumor vessel tortuosity and vessel thinning, and improved vessel function and blood flow. The tumor vessel stabilization effect of the PI3K inhibitor HS-173 also decreased the number of metastatic lung nodules in vivo metastasis model. Furthermore, HS-173 improved the delivery of doxorubicin into the tumor region, enhancing its anticancer effects. Mechanistic studies suggested that PI3K inhibitor HS-173-induced vessel normalization reflected changes in endothelial Notch signaling. Taken together, our findings indicate that vessel normalization by PI3K inhibitors restrained tumor growth and metastasis while improving chemotherapy by enhancing drug delivery into the tumor, suggesting that HS-173 may have a therapeutic value as an enhancer or an anticancer drug.

Introduction

Tumor angiogenesis, the formation of new blood vessel, plays an essential role in tumor progression [1], [2]. Extensive angiogenesis in tumors is accompanied by a defective vascular architecture, leading to increased vascular permeability, hypoxia, and high interstitial fluid pressure. Cancer growth strictly depends on the expansion of the host vasculature, which not only supplies oxygen and nutrients to the tumor tissue, but also provides cancer cells with a metastatic route for colonizing distant organs [3], [4], [5]. Therefore, angiogenesis represents a critical process during tumor initiation and malignant progression, and is a target of anticancer drugs. Some studies have shown that inhibition of angiogenesis suppresses metastasis and tumor growth [6], [7], whereas others have reported that it is associated with enhanced intratumoral hypoxia, increased local tumor invasion, and frequency of metastasis [7], [8]. Indeed, the benefits of antiangiogenic agents are transient, and the initial response is often followed by the establishment of resistance and escape mechanisms, leading to tumor relapse [7], [9]. These observations accentuate the need for a more comprehensive understanding of the biological processes that underlie tumor vascularization and set the stage for additional angiogenesis-targeted therapies.

Compared with normal vessels, tumor vessels are heterogeneous and tortuous, branch chaotically, and have an uneven vessel lumen. They also frequently lack pericyte coverage and show an abnormal basement membrane, resulting in vascular instability and altered permeability. These vessel abnormalities generate a promalignant microenvironment that can facilitate the dissemination of cancer cells through leaky vessels, thus causing poor response to therapy [10]. In addition, normal vessels are composed of a monolayer of interconnected adherent endothelial cells (ECs), whereas tumor vessel ECs can detach from the basement membrane and vessels, resulting in pericyte loss [11]. Namely, the structural stability of vessels is dependent on both the distribution of ECs and pericyte coverage [12], [13], factors that are important in promoting vessel normalization.

These observations raise the question of whether tumor vessel normalization offers an alternative therapeutic strategy for reducing metastatic spread and enhancing tumor responses to chemotherapy and radiotherapy [14]. Recent studies have typically used ‘vessel blocking’ to describe a situation in which the total number of immature and mature vessels is reduced, and ‘vessel normalization’ in case where only immature vessels are removed and the remaining vessels become more normal and mature [15]. Thus, in the process of vessel normalization, the architecture of the remaining vasculature is largely restored, thereby reducing vessel permeability and improving tumor blood flow [15], [16], [17].

The effects of vessel normalization may be even more important for intratumoral delivery of drugs [18]. Several preclinical and clinical studies have indicated that antiangiogenic drugs lead to vessel normalization in addition to causing anti-vascular effects [19], [20]. One example is bevacizumab, which is mainly given in combination with chemotherapy. The beneficial effect of this combination therapy is attributable to vessel normalization and the consequent increase in tumor uptake of co-delivered chemotherapeutic agents [21]. However, most anti-angiogenic drugs, such as vascular endothelial growth factor receptor (VEGFR) inhibitors, have failed to optimize drug-dose and interval for the combination, such that the drug induces metastasis through increased vessel leakiness and destruction of ECs [22].

A number of previous studies have reported that phosphoinositide 3-kinase (PI3K) signaling affects tumor angiogenesis both directly and indirectly [23]. PI3K activation also upregulates VEGF and promotes vessel formation [24]. Thus, inhibition of the PI3K pathway suppresses tumor cells and affects the tumor stroma, including the vasculature. Indeed, targeting PI3K signaling has been shown to induce antiangiogenic activity in preclinical tumor models [25], [26], [27], [28]. In addition, PI3K inhibitors block neovascularization and downregulate VEGF, and also inhibit tumor growth [29], [30], [31]. Recently, the PI3K inhibitor BEZ235 has been shown to increase blood perfusion in normalized tumor vessels, resulting in an antiproliferative effect by counterbalancing the increased tumor growth [32]. Similarly, the EGFR inhibitor erlotinib has shown improved delivery of cisplatin and synergistic inhibition of tumor growth by increasing perfusion. These previous studies suggest that these drugs produce a more durable induction of vascular normalization. However, only a limited number of studies have investigated the effects of PI3K inhibitors on vessel normalization and its mechanisms are not well understood.

In this study, we investigated the potential of HS-173, a novel PI3K inhibitor, as well as BEZ235 to inhibit tumor growth by inducing vascular normalization. We found that HS-173 and BEZ235 caused vessel normalization in tumors, inhibiting vascular leakage, improving capillary integrity, and increasing vessel density. This was particularly notable for HS-173 in combination therapy, which increased blood perfusion and led to delayed tumor growth. Therefore, vessel normalization by HS-173 could be a novel therapeutic strategy for inhibiting tumor growth by improving drug delivery to the tumor.