3.2.1. Angiogenesis

Angiogenesis describes the process of vessel formation and is crucial for tumor growth and metastasis. Among angiogenesis markers, VEGFR2 and αvβ<sup>3</sup> integrin are the most addressed targets. VEGFR2 also known as KDR is an endothelium-specific receptor, that is highly expressed on tumor-associated endothelial cells [105]. Activation of the VEGFR2 pathway triggers multiple signaling cascades that result in endothelial cell survival, mitogenesis, migration, differentiation, and alterations in vascular permeability [103]. Moreover, overexpression of VEGFR2 has been linked to tumor progression and poor prognosis in several tumors [106]. Integrins are transmembrane receptors that are expressed on endothelial and tumor cells. They activate signaling cascades that regulate gene expression, cytoskeletal organization, cell adhesion and cell survival. This facilitates tumor growth, invasion and metastasis [107]. In particular, αvβ<sup>3</sup> integrin expression is low on endothelial cells under normal conditions, but is elevated during tumor angiogenesis [108–110]. Another angiogenesis related marker is Endoglin, a transmembrane glycoprotein expressed in proliferating endothelial cells such as tumor endothelial cells [111]. It is a component of the transforming growth factor beta receptor complex involved in cell proliferation, differentiation and migration [111].

MB functionalized with ligands against VEGFR2, integrin and other angiogenesis markers have been successfully tested for molecular US imaging (Table 1). Moreover, the CEUS signal of the targeted MB could be correlated to the level of angiogenesis marker expression in the tissue. For example, CEUS signal using VEGFR2-targeted MB increased from hyperplasia to ductal carcinoma in situ and invasive breast cancer compared to normal tissue [46]. This was additionally confirmed by histological analysis.

During tumor angiogenesis, multiple endothelial markers are overexpressed [112]. These markers could be good targets for cancer detection. In this respect, E-selectin [86,87], secreted frizzled related protein 2 (SFRP2) [113], B7–H3 [54], nucleolin [55] and thymocyte differentiation antigen 1 (Thy1) [56] were tested for cancerous tissue detection and showed promising results. Since angiogenesis is an early event in tumor development [114], angiogenesis targeted MB should be able to detect tumors at early stages. Indeed, ovarian cancer at an early stage was sensitively detected using αvβ<sup>3</sup> integrin-targeted MB [27]. Also, VEGFR2-targeted UCA were able to visualize breast cancer tumors as small as 2 mm in diameter [44,46] and pancreatic ductal adenocarcinoma lesions smaller than 3 mm in diameter [47]. Interestingly, a study showed that smaller breast cancer xenografts express the highest amount of VEGFR2, while with increasing tumor size the expression decreases [44]. Thus, current results suggest that VEGFR2 targeted MB are promising UCA for early tumor detection, though more research should be done.

Since angiogenesis plays an important role in tumor growth, it is not surprising that multiple anti-cancer drugs target angiogenesis. The idea is to stop tumors from developing new blood vessels and hopefully shrink them by cutting the nutrient and oxygen supply. CEUS imaging using targeted MB can provide information on the angiogenesis profile of the tumor and assess antiangiogenic therapy effects. Several groups showed successful monitoring of antiangiogenic therapy using VEGFR2-targeted MB [29,31,35,45,49,50]. In all cases, a clear decrease of VEGFR2-targeted MB accumulation was observed after treatment compared to untreated controls. These findings were further confirmed with immunohistochemistry analysis. Moreover, other therapies such as gemcitabine [35], nilotinib [45], and carbon ion treatment [28] were monitored using CEUS with angiogenesis targeted MB. Similarly, as for antiangiogenic therapy monitoring, CEUS was correlated to the target marker expression according to immunohistochemistry analysis. Hence, CEUS using angiogenesis targeted MB seems to be a promising tool for non-invasive antitumor therapy monitoring.

Furthermore, by combining functional and molecular US imaging, vascular responses in tumors can be comprehensively characterized. This is important since the percentage of angiogenic vessels indicating angiogenic activity – cannot be assessed solely from the information on VEGFR2 bound MB. For, example, Palmowski and co-workers showed that VEGFR2 and αvβ<sup>3</sup> integrin-targeted MB bound less after administration of a matrix metalloproteinase (MMP) inhibitor, which was in line with immunohistochemical analysis. However, when normalizing the molecular marker expression to vascular density no change in the percentage of angiogenic vessels was visible indicating that the MMP inhibitor did not decrease angiogenesis but induced a general decrease in vascularization [29]. To unravel these effects by US, Bzyl et al. injected long-circulating UCA (BR38) to derive functional information on vascularization and relative blood volume and VEGFR2-targeted MB (BR55) to assess angiogenesis [39]. Two breast cancer models with different aggressiveness and angiogenic activity were evaluated. The results showed that the more aggressive tumor model (MDA-MB-231) had the higher total expression of VEGFR2 compared to MCF-7 according to VEGFR2-targeted US measurements and immunohistochemistry. Also, the normalization of the molecular imaging data to the relative blood volume confirmed the higher angiogenic activity of the MDA-MB-231 model. A few years later Baetke et al. showed that functional and molecular US imaging can even be performed in one examination using only the targeted MB [49]. Here, antiangiogenic therapy effects were monitored in squamous cell carcinomas using BR55. It was shown, that the first-pass analysis of VEGFR2-targeted MB was not strongly affected by the targeting, and vascularization results were comparable to those obtained with non-targeted MB. Hence, in the early binding phase, functional information could be obtained and at the late phase, the molecular angiogenesis profile of the tumors could be assessed (Figure 5). Combining functional and molecular US in one examination would reduce measurement time and the need for multiple injections and multiple formulations of MB.

In summary, all these pre-clinical studies using angiogenesis-targeted MB showed promising results confirming that the targeted UCA can be used for angiogenesis profiling in various tumors. Most research has been done using VEGFR2-targeted MB, especially using the clinical-grade contrast agent BR55. These MB can detect tumors at early stages and can be used for antiangiogenic therapy monitoring combining functional and molecular US. BR55 is now evaluated in clinical trials and the initial results are summarized in Section 3.2.5.

**Figure 5.** CEUS images of treated (top) and untreated tumors (bottom). Left side: early vascular phase with VEGFR2-targeted MB as a functional imaging biomarker; Right side: late phase of VEGFR2-specific binding with the targeted MB as a molecular imaging biomarker 8 min after contrast injection. Reproduced with permission from [51]. Copyright Eschbach et al., 2017.
