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

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

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

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

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

Tumor-positive margin and IVIS image after partial tumor resection.

<p>(A) Histological Image of the resected tumor from mouse S5 with tumor-positive margin. (B) IVIS image of three different mice two weeks after receiving a tumor resection. The IVIS image from mouse S4 shows a positive bioluminescent signal in the previous tumor area (ROI 1) two weeks after receiving a tumor resection. In this mouse, the histological exam showed tumor-free margin from the resected tumor. The IVIS image of positive bioluminescent signal in the previous tumor area near the tail is due to skin shifting after stitching. In the IVIS image of mouse S5 no residual tumor was observed two weeks after receiving a tumor resection but the histology exam of the resected tumor showed a positive tumor margin.</p

CTC dynamics change after tumor pressure.

<p>(A) Profile plot of CTC detection rates (in CTCs/minute) measured weekly from six mice inoculated with breast cancer cells (MDA-MB-LUC2-GFP) during the eight weeks after tumor inoculation. Pressure was applied at week 2. (B) Profile plot of the average number of CTC signals per minute and average tumor volume during the same eight weeks. Values and error bars represent the averages and SDs of CTC counts from n = 6 mice. (C) Individual tumor volumes from six mice after tumor inoculation. Pressure was applied at week 2 after tumor inoculation. (D) Profile plot of average number of CTC signals per minute from 60 min before, 15 min during, and 120 min after removing pressure provided by a cylindrical 400 g weight with 10-mm diameter. (E) Image of the tumor after 15 minutes of compression using digital pressure-controller software (Loadstar Sensor, DI-100).</p

Change in CTC dynamics after surgery.

<p>(A) Profile plot of CTC detection rates (in CTCs/minute) measured weekly from six mice inoculated with breast cancer cells (MDA-MB-LUC2-GFP). Surgery was conducted at Week 2 after tumor inoculation. (B) Profile plot of the average number of CTC signals per minute and average tumor volume during eight weeks from n = 5 mice after excluding the mouse which received partial tumor resection. Values and error bars represent the averages and SDs of CTC counts from n = 5 mice. (C) Individual tumor volumes from six mice. Surgery was performed at week 2 after tumor inoculation. (D) Profile plot of the average number of CTC signals per minute from 60 min before and 120 min after the surgery.</p

Change in CTC dynamics after punch biopsy.

<p>(A) Profile plot of CTC detection rates number of CTCs per a minute measured weekly from six mice inoculated with breast cancer cells (MDA-MB-LUC2-GFP) during the eight weeks after tumor inoculation. Punch biopsy was performed at week 2 after tumor inoculation. (B) Profile plot over time of the average number of CTC signals per minute and average of the tumor volume during the same eight weeks. Values and error bars represent the averages and SDs of CTC counts from n = 6 mice. The average of CTC rate per 8 weeks was calculated for five mice at week 3, three mice at week 5 and two mice at week 7. (C) Individual tumor volumes from six mice. Punch biopsy was performed at week 2 after tumor inoculation. (D) Profile plot of the average number of CTC signals per minute from 60 min before and 120 min after the punch biopsy, showing a 1.82-fold increase in the CTC-detection rate (P = 0.02). (E) Image of the tumor after a punch biopsy.</p