Best-fit values described models for <i>ffc-</i>TICs.

<p>Best-fit values described models for <i>ffc-</i>TICs.</p

Best-fit values described models for wash out of contrast from non-tumor ROI.

<p>Best-fit values described models for wash out of contrast from non-tumor ROI.</p

Best-fit values described nonlinear curves for wash out of contrast.

<p>Best-fit values described nonlinear curves for wash out of contrast.</p

Average video pixel intensity increased with increased microbubble dose.

<p>Significantly higher video pixel intensity was observed in tumors when animals received 5 × 10⁷ SFRP2-targeted microbubbles compared to 5 × 10⁶ SFRFP2-targeted microbubbles. N = 10 animals.</p

Time-Intensity Curves (TICs) generated from a representative animal that received both SFRP2-targeted, and IgY-targeted control contrast.

<p><b>(A)</b> SFRP2-targeted contrast: solid blue line represents intensity within the tumor ROI, dotted black line represents intensity within a non-tumor ROI, red filled circles represent the difference between the tumor and non-tumor ROI intensities, which we term ‘free-flowing-corrected’ TIC (<i>ffc</i>-TIC). <b>(B)</b> IgY-targeted contrast: solid green line represents intensity within the tumor ROI, dotted black line represents intensity within a non-tumor ROI, purple open circles represent the difference between the tumor and non- tumor ROI intensities, which we term ‘free-flowing-corrected’ TIC (<i>ffc</i>-TIC). <b>(C)</b> The wash out of contrast from tumor ROI was modeled by one-phase exponential decay for IgY-targeted contrast (raw data, open green circles; green line, best-fit model), and by a plateau followed by one-phase exponential decay for SFRP2-targeted contrast (raw data, solid blue circles; blue line, best-fit model). <b>(D)</b> The difference between the models depicted in panel (C) was plotted with open black triangles. Note the maxima between 6–10 minutes. <b>(E)</b> The <i>ffc</i>-TICs for SFRP2, and IgY-targeted contrast were fitted to curves. A one-phase association model (grey line) fit the wash in portion of the <i>ffc</i>-TIC for SFRP2-targeted contrast (red filled circles), and for IgY-targeted contrast (open purple circles). The wash out of SFRP2-targeted <i>ffc</i>-TIC was best fit with a linear regression model (blue line), while the wash out of IgY-targeted <i>ffc</i>-TIC was best fit with a one-phase exponential decay model (green line). <b>(F)</b> The best-fit model for the IgY-targeted <i>ffc</i>-TIC was subtracted from the best-fit model for SFRP2-targeted <i>ffc</i>-TIC, and was plotted (red filled circles with red line). This produced a TIC representing the signal intensity within the tumor ROI that could be attributed to binding of contrast specifically mediated by the SFRP2 antibodies used to formulate the SFRP2-targeted contrast. N = 5 animals.</p

Average video pixel intensity increased with higher levels of NeutrAvidin^™ labeling.

<p>Significantly higher tumor video pixel intensity was observed when SFRP2-targeted contrast was created using 10-fold molar excess of maleimide-activated NeutrAvidin^™ compared to 3-fold molar excess. N = 8 animals.</p

The relationship between microbubble acoustic signal, and log compressed video pixel intensity.

<p><b>(A)</b> The acoustic signal from microbubbles in arbitrary acoustic units (AU) was log compressed from a dynamic range of 80 dB to an 8-bit range (0–255) of video pixel intensities (VI). Given video pixel intensities (VI), log decompression with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174281#pone.0174281.e001" target="_blank">Eq 1</a> was used to estimate the original acoustic signal in AU, and was plotted to show their non-linear relationship over the entire 8-bit range. <b>(B)</b> A linear relationship existed between video pixel intensities (VI), and their corresponding acoustic signals (AU), when VI was < 20. This linear relationship represented a range of acoustic signals limited to ~ 0.025–0.055 AU. <b>(C)</b> The ratio of any two video pixel intensities (VI) was not related linearly to the ratio of their estimated acoustic signals. (D) However, the ratio between log-decompressed acoustic signals (<i>AU</i>_<b>2</b> ÷ <i>AU</i>_<b>1</b>) was related linearly to Δ VI = (VI2 –VI1). Computing ΔVI resulted in a metric that was proportional to the ratio of the original acoustic signals: a natural consequence of the Law of Exponents. Linear regression provided the best-fit equation: (<i>AU</i>_<b>2</b> ÷ <i>AU</i>_<b>1</b>) = 0.0529 × Δ<i>VI</i> + 0.9455, with R^<b>2</b> = 0.992 describing the relationship between ΔVI, and (<i>AU</i>_<b>2</b> ÷ <i>AU</i>_<b>1</b>) when ΔVI < 20.</p

Ultrasound molecular imaging of animal receiving SFRP2-targeted and control IgY-targeted contrast.

<p>A white dashed line outlines tumors. The contrast-specific signal (green) was superimposed over the b-mode image (grey). At 30 seconds, average video pixel intensity was similar between control and SFRP2-targeted contrast. The contrast-specific video intensity was retained in tumors at much higher levels when using the SFRP2-targeted contrast compared to the IgY-targeted contrast.</p

Verifying performance of optimized molecular imaging reagents, and protocol <i>in vivo</i>.

<p>Non-identical superscripts indicate a statistically significant difference in means. Both IgY-targeted (a, p = 0.03), and SFRP2-targeted (c, p < 0.001) contrast produced significantly higher video pixel intensities in tumor ROI compared to peri-tumoral regions (b). SFRP-2 targeted contrast (c, p < 0.001) produced significantly higher signal intensity in tumor ROI than the control IgY-targeted contrast (a). Average video pixel intensities were corrected for free flowing contrast by subtracting the post-destruction VI from the pre-destruction VI as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174281#pone.0174281.g002" target="_blank">Fig 2B</a> for the ‘image—destroy—image’ method. N = 10 animals.</p

Modified microbubble contrast agents were not retained at significant levels in nonmalignant vasculature.

<p>B-mode images (black and white) are shown overlaid with CPS-mode images (green). CPS-mode is sensitive to ultrasound signal typically produced by microbubbles oscillating within an ultrasound field. (A) In the absence of ultrasound contrast agent (no contrast) there was no CPS-mode signal within the region of interest (dotted rectangle) outside of the tumor margins. Tissue artifacts generated the CPS-mode signal observed in the absence of contrast agent. Contrast agent freely flowing through both tumor and non-tumor vasculature generated CPS-mode signal throughout the field of view (panel A, perfusion, middle frames) with either streptavidin-coated (control, upper frames) or SFRP2- targeted ultrasound contrast agent (lower frames). No signal remained within the region of interest drawn outside of the tumor margins after allowing all freely flowing contrast agent to be cleared from the vasculature, while SFRP2 specific signal was retained within the tumor margins. (B) Modified microbubble contrast agents were not retained within kidney vasculature. Freely flowing streptavidin-loaded microbubbles (panel B, control, upper frames) or SFRP2 - targeted microbubbles (panel B, lower frames) were allowed to clear from the vasculature prior to three-dimensional molecular imaging. Single frames are shown from two different animals (animal 1 or animal 2). The dotted oval region of interest represents the location of the kidney (K) and there was no significant difference in average pixel intensity after injection of either streptavidin-loaded or SFRP2 -targeted microbubbles. In contrast, the liver (L) retained both modified microbubble contrast agents to a high degree. White scale bars in panels A and B represent 5 mm.</p