Oxygen Microbubbles Improve Radiotherapy Tumor Control in a Rat Fibrosarcoma Model – A Preliminary Study

Cancer affects 39.6% of Americans at some point during their lifetime. Solid tumor microenvironments are characterized by a disorganized, leaky vasculature that promotes regions of low oxygenation (hypoxia). Tumor hypoxia is a key predictor of poor treatment outcome for all radiotherapy (RT), chemotherapy and surgery procedures, and is a hallmark of metastatic potential. In particular, the radiation therapy dose needed to achieve the same tumor control probability in hypoxic tissue as in normoxic tissue can be up to 3 times higher. Even very small tumors (<2–3 mm3) comprise 10–30% of hypoxic regions in the form of chronic and/or transient hypoxia fluctuating over the course of seconds to days. We investigate the potential of recently developed lipid-stabilized oxygen microbubbles (OMBs) to improve the therapeutic ratio of RT. OMBs, but not nitrogen microbubbles (NMBs), are shown to significantly increase dissolved oxygen content when added to water in vitro and increase tumor oxygen levels in vivo in a rat fibrosarcoma model. Tumor control is significantly improved with OMB but not NMB intra-tumoral injections immediately prior to RT treatment and effect size is shown to depend on initial tumor volume on RT treatment day, as expected

Experimental procedures.

<p>a) Schematic of the experimental set-up used for in-vivo hypoxia modulation measurements using the Zenascope system. b) Schematic of tumor volume assessment via B-mode ultrasound imaging. Two cross-sectional images were acquired and lengths a, b and c were used to calculate tumor volume. c) Radiotherapy pre and post-imaging experimental protocol.</p

In vitro oxygen microbubble characterization.

<p>a) Measured oxygen microbubble size distribution, displayed with a diameter bin size of 0.032 μm, as mean ± standard deviation (gray area) from 3 independent samples; b) Measured change in oxygen % saturation in vitro after 300 μL OMB (n = 3) or NMB (n = 3) injection into 70 mL partially degassed water (p<0.05).</p

Oxygen microbubbles improve radiotherapy tumor control in a rat fibrosarcoma model – A preliminary study

<div><p>Cancer affects 39.6% of Americans at some point during their lifetime. Solid tumor microenvironments are characterized by a disorganized, leaky vasculature that promotes regions of low oxygenation (hypoxia). Tumor hypoxia is a key predictor of poor treatment outcome for all radiotherapy (RT), chemotherapy and surgery procedures, and is a hallmark of metastatic potential. In particular, the radiation therapy dose needed to achieve the same tumor control probability in hypoxic tissue as in normoxic tissue can be up to 3 times higher. Even very small tumors (<2–3 mm³) comprise 10–30% of hypoxic regions in the form of chronic and/or transient hypoxia fluctuating over the course of seconds to days. We investigate the potential of recently developed lipid-stabilized oxygen microbubbles (OMBs) to improve the therapeutic ratio of RT. OMBs, but not nitrogen microbubbles (NMBs), are shown to significantly increase dissolved oxygen content when added to water in vitro and increase tumor oxygen levels in vivo in a rat fibrosarcoma model. Tumor control is significantly improved with OMB but not NMB intra-tumoral injections immediately prior to RT treatment and effect size is shown to depend on initial tumor volume on RT treatment day, as expected.</p></div

Individual datapoints for radiotherapy tumor control times (in days) stratified by matched initial tumor size for each treatment group, showing RT effect size depends on initial tumor volume.

<p>Individual datapoints for radiotherapy tumor control times (in days) stratified by matched initial tumor size for each treatment group, showing RT effect size depends on initial tumor volume.</p

Change in tumoral oxygenation with intra-tumoral injection of OMB or NMB.

<p>The time to peak was found to be 97 s after injection on average, and the OMB-induced increase in tumoral oxygenation lasted for over 18 min on average (our protocol’s maximum 1 h experiment time meant that we could not wait for a complete return to baseline in some cases). A) Average peak change in tumoral hemoglobin saturation after OMB or NMB administration (n = 4/group). B) Individual data points showing pre- and post-injection values. This demonstrates baseline hypoxia in all tumors (0–83% hemoglobin saturation across all 8 tumors).</p

(Color online) example of acoustic angiography maximum intensity projections around tumors (tumor size denoted with dashed yellow lines) in a rat fibrosarcoma allograft, with tortuous angiogenesis extending beyond the tumor margins (red arrows).

<p>The small tumor (a) is also shown to be more enhanced, denoting its higher perfusion compared to the larger tumor (b).</p

Tumor control time comparison between RT groups.

<p>OMB significantly improve RT outcome, whereas NMB as controls do not (n = 6 per group). Box-and-whisker plots show all data from the three radiotherapy treatment groups.</p

Plot of gain in tumor control time against initial tumor volume, the confounding variable.

<p>An on/off effect (threshold) is observed around 0.5 cm³ initial tumor volume. Below this size, tumors are controlled for 31 days with RT alone. Above this size, tumors are large enough that RT alone cannot control them for 31 days, and therefore, OMB administration can provide a substantial improvement in tumor control time. Additionally, the benefit offered by OMB administration diminishes as initial tumor volume exceeds ~2 cm³. Given our study design, the slope describing the inverse relationship between improvement in tumor control and initial tumor volume (above the threshold value of 0.5 cm³) could in theory guide optimal OMB dosing with respect to tumor volume (confounder) in order to maximize tumor control benefit for a given RT dose.</p

A single oxygen microbubble administration alone does not influence tumor control.

<p>No significant difference was found between the no treatment and oxygen microbubble group in the absence of any radiotherapy (n = 4 per group). Box-and-whisker plots represent all data from the No treatment and OMB alone controls.</p