Targeting Artificial Tumor Stromal Targets for Molecular Imaging of Tumor Vascular Hypoxia

<div><p>Developed and tested for many years, a variety of tumor hypoxia detection methods have been inconsistent in their ability to predict treatment outcomes or monitor treatment efficacy, limiting their present prognostic capability. These variable results might stem from the fact that these approaches are based on inherently wide-ranging global tumor oxygenation levels based on uncertain influences of necrotic regions present in most solid tumors. Here, we have developed a novel non-invasive and specific method for tumor vessel hypoxia detection, as hypoxemia (vascular hypoxia) has been implicated as a key driver of malignant progression, therapy resistance and metastasis. This method is based on high-frequency ultrasound imaging of α-pimonidazole targeted-microbubbles to the exogenously administered hypoxia marker pimonidazole. The degree of tumor vessel hypoxia was assessed in three mouse models of mammary gland carcinoma (4T1, SCK and MMTV-Wnt-1) and amassed up to 20% of the tumor vasculature. In the 4T1 mammary gland carcinoma model, the signal strength of α-pimonidazole targeted-microbubbles was on average 8-fold fold higher in tumors of pimonidazole-injected mice than in non-pimonidazole injected tumor bearing mice or non-targeted microbubbles in pimonidazole-injected tumor bearing mice. Overall, this provides proof of principle for generating and targeting artificial antigens able to be ‘created’ on-demand under tumor specific microenvironmental conditions, providing translational diagnostic, therapeutic and treatment planning potential in cancer and other hypoxia-associated diseases or conditions.</p></div

High-frequency ultrasound imaging of targeted-microbubbles detects tumor vessel hypoxia.

<p>Representative image and quantified data of anti-pimonidazole labeled microbubbles (MBα-pimo) bound in perfused hypoxic tumor vasculature without pimonidazole injection <b>(A)</b>, and with pimonidazole injection <b>(B)</b> in 4T1 tumor bearing mice. Top image shows the signal before the burst sequence and the bottom image shows after the burst sequence <b>(A, B)</b>. <b>(C)</b> Quantified data of different experimental conditions using targeting and non-targeting microbubbles (as indicated). <b>D)</b> Summary of quantitated data statistically analyzed represented as mean ± SEM, ^#p < 0.05, versus non-targeting MB, MBα-pimo without pimonidazole injection, and MBα-pimo in muscle tissue (ANOVA post-hoc Holm-Sidak).</p

3D modeling of MBα-pimo distribution in mammary gland carcinoma.

<p><b>A)</b> Single slice images taken from a 3D imaging sequence in B-mode <i>(left)</i>. Single slice images taken from a 3D imaging sequence depicting the differential targeted expression (d.T.E) <i>(right)</i>. <b>B)</b> Three-dimensional contrast projection of 3D stack image data from hypoxia targeted, MBα-pimo, contrast signal collected in a rear-limb 4T1 tumor. Images (0.152mm/slice) generated using Visualsonics imaging system and post-processed using the Huygens essential software.</p

Vascular hypoxia in murine breast carcinomas and normal tissue.

<p>Immunofluorescence analysis of hypoxemia in 4T1 mammary gland carcinoma. <b>(A)</b>, 4T1 tumor tissue is stained for tumor vessels (CD31; red). (<b>B)</b> Tumor hypoxia (pimonidazole; green) is co-localized (white) in relation to microvasculature in 4T1 tumor tissue <b>(C)</b>. Quantification of overall tumor vessels <b>(D)</b>, hypoxia <b>(E)</b>, and hypoxic tumor vessels <b>(F)</b> in 4T1, SCK and MMTV-Wnt-1 carcinomas. Immunofluorescence analysis of vasculature (CD31; red) and hypoxia (pimonidazole; green) in non-diseased kidney <b>(G)</b>, spleen <b>(H)</b> and liver <b>(I)</b> indicates a lack of global and vessel hypoxia in normal tissue.</p

Schematic of anti-pimonidazole targeted-microbubbles (MBα-pimo) with ultrasound imaging for detection of vascular hypoxia.

<p><b>(A)</b> Illustration showing the differential distribution of MBα-pimo in well-oxygenated tumor endothelium (red) compared to hypoxic tumor endothelium (blue) during imaging and intervention by ultrasound. <b>(B)</b> Representative quantification graphic of MBα-pimo where the binding occurs over a 5 minute window after IV injection followed by a data collection period of contrast signal, a single ultrasound pulse to burst bound and free MBα-pimo, and a final data collection during the immediate reperfusion window. Subsequently, the difference in signal from the steady state prior to microbubble burst (‘pre’) and following burst (‘post’) can be calculated. This differential targeted expression (d.T.E.; linear, a.u.) represents the relative amount of bound MBα-pimo and indirectly indicates the location and amount of vascular hypoxia within the tumor (x-axis scale not linear).</p

Pimonidazole targeting microbubbles.

<p><b>(A)</b> A graphic representation of the microbubbles and conditions used. <i>Left</i>, unlabeled microbubbles (MB); middle, pimonidazole-targeting MB (MBα-pimo); and <i>right</i>, MBα-pimo without pimonidazole present in the circulation. <b>(B)</b> MBα-pimo binds hypoxic 2H11 endothelial cells only in the presence of pimonidazole. MBα-pimo does not bind endothelial cells (<i>left</i>), unless pimonidazole is added (<i>middle</i>). (<i>right</i>), MBα-pimo binding to the cell surface of hypoxic endothelial cells, magnification 40X.</p