found followed by onset of hypoxia (pO
,10 mmHg). It should be
noted that the USPIO-based blood volume assessment may
overestimate the values in tumors because of their leakiness
compared to normal tissues . The observations from
histological experiments were in agreement with the imaging
observations. The rapamycin-induced decrease in CD31 staining
was found to be in agreement with imaging experiments where a
loss in microvessel density was found. However, there was a small
but non-significant decrease in staining of aSMA which reflects the
retention of the integrity of the pericyte coverage of the tumor
vasculature after rapamycin administration. These results indicate
that rapamycin treatment pruned immature blood vessels rather
than mature blood vessels. It is expected that these changes in
tumor microvasculature can cause improvement of blood flow, a
phenomenon known as vascular normalization. The transient
increase in the pO
by rapamycin treatment can be attributed to
the increased blood flow in the tumor, which was demonstrated by
a 40% increase in tumor initial uptake of Gd-DTPA 2 days after
rapamycin treatment in the DCE-MRI study.
The identification of transient improvements in tumor oxygen-
ation 2 days after rapamycin treatment provides an opportunity
for chemoradiation modalities where radiation therapy can be
timed to take advantage of increases in tumor pO
improved response . The results in the present study show
enhancement in tumor radioresponse by rapamycin treatment
(Figure 6). This data suggests that the transiently increased level of
median tumor pO
in rapamycin treated mice compared to the
day matched control group may be responsible for the observed
effect of radioresponse with combination treatment. The relatively
smaller effect of radiation with rapamycin (additive), in contrast
with the observed synergistic effect of radiation with sunitinib in
the same tumor xenograft , may be explained in terms of the
relatively smaller magnitude difference in tumor pO
cin treated group to the day matched control (,2 mm Hg)
compared to the greater difference in tumor pO
treated group to the control (,5.5 mm Hg). The significant
synergy with mTOR inhibitors including rapamycin and radiation
reported by Shinohara et al  may point out the characteristic
influences of the microenvironment of each tumor type as pointed
out in other studies where the synergy was attributed only to
rapamycin targeting the enhanced activity of signaling pathways
controlled by mTOR in the host endothelial cells . Recent
studies with a dual inhibitor of the PI3K and mTOR pathway
found that the period of vascular remodeling is relatively more
sustained than that observed with anti-angiogenic drugs resulting
in substantial therapeutic gain . These studies point to the
importance of longitudinally monitoring such changes to realize
maximal efficacy in combined chemo-radiation treatments.
Imaging studies of the tumor microenvironment can establish a
strategy in preclinical models to identify an optimal treatment
schedule to realize enhanced response to combination treatments.
In summary, results from the current study show that molecular
imaging techniques provide an opportunity to serially monitor
changes in tumor physiology non-invasively and quantitatively and
identify subtle physiological changes in response to rapamycin
treatment. Therefore these techniques have the ability to provide
valuable non-invasive biomarkers which predict treatment out-
come and also identify temporal windows where radiation therapy
can be advantageously combined to elicit improved response.
Conceived and designed the experiments: KS SM VP JSG JBM MCK.
Performed the experiments: KS SM HY VP. Analyzed the data: KS SM
VP JSG JBM MCK. Contributed reagents/materials/analysis tools: ND
SS JPM. Wrote the paper: KS SM MCK.
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Rapamycin Improves Tumor Oxygenation
PLOS ONE | www.plosone.org 8 November 2012 | Volume 7 | Issue 11 | e49456