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<p>Background: PD-L1 (Programmed Death Ligand 1) is an immune checkpoint molecule
that is commonly expressed on the surface of cancer cells. When it interacts with
its receptor – the PD-1 molecule, which is commonly expressed on the surface of immune
cells, such as T-cells, it will then deliver a negative signal which in turn inactivates
the T-cell function, depresses the T-cell expansion, and dampens the overall tumor
response. Anti-PD-L1 antibody blocks the direct interaction between the PD-L1 and
PD-1 molecules, thus inhibits the PD-L1 signaling pathways, enabling the immune system,
and hence the anti-tumor immunity, to eliminate the cancer cells. We refer to these
mechanisms as the Immune Checkpoint Blockade (ICB). </p><p>Methods and Materials:
In the current study, we report the effects of combination treatment of radiation
and ICB on tumor metastasis. A single radiation dose of 10 Gy was used to irradiate
the dorsal fat pad region of mice, into which the E0771-luc breast cancer cells were
injected. For the ICB treatment, anti-PD-L1 antibody was used. We established a spontaneous
metastatic model using E0771-luc breast cancer cells. As the tumor grew, primary tumor
growth was monitored using calipers. We then examined the metastatic lung lesions
by using In-Vivo Imaging System (IVIS), the signals from IVIS were then quantified
in terms of total photon flux (photons/second). An India-Ink Assay was also employed
to further verify the lung mass formations. In addition, variation of primary tumors’
hemoglobin saturation levels and total hemoglobin levels were recorded before and
after treatments using zenascope, for the sake of assessing the vascular oxygenation
and vascularity information, respectively.</p><p>Principal Findings: Data from calipers’
measurement showed that the RT-alone group did not show any effects on tumor growth.
Tha anti-PD-L1-alone group showed a slightly delayed tumor growth. The combination
treatment showed that 3 out of 5 mice showed tumor growth delay, while the tumor regrew
after 20 days. Furthermore, survival curves indicated that there is no significant
difference among all groups, indicating that radiation treatment or anti-PD-L1 antibody
or the combination of both treatments did not affect the time at which the mice reach
their endpoints (tumor volume ≥ 1500mm3). In addition, data from the IVIS indicated
that the total photon flux emitted from primary tumors varied dramatically among experimental
subjects within the same group. There were also extremely low or no luciferase signals
from the lung. Statistically, two-way ANOVA for IVIS data showed that there are no
significant differences between the RT-alone or anti-PD-L1-alone or RT + anti-PD-L1
group and the control group, for both primary tumors and lung tissue, suggesting that
all kinds of treatments used in the current study neither helped eliminate the primary
tumor cells nor reduced the burden of metastatic cancer cells in lungs, compared to
the control group. Interestingly, results from the India-ink Assay showed that grossly
visible lung nodules were not observed in all lungs of the mice, suggesting that the
primary tumors in the dorsal fat pad region did not result in grossly visible lung
metastases in any groups. Furthermore, analysis for the Zenascope data showed that
there was a gradual increase in Hb-Sat(%) in mice for the control group, while a gradual
decrease in Hb-Sat(%) for the anti-PD-L1-alone group. The RT-alone group did not show
a clear response of change in Hb-Sat. For the combination treatment group, 3 out of
4 mice demonstrated a relatively flat response of change in Hb-Sat(%). Lastly, the
total Hb levels in the control group, the anti-PD-L1-alone group and the combination
treatment group remained relatively stable over the treatment time. For the RT-alone
group, 3 out of 5 mice showed almost no changes, while the other two demonstrated
a huge increase in total Hb levels on day 0, and day 2, respectively, but the levels
went back to almost the pre-treatment values after day 3. </p><p>Conclusions: The
combination treatment of 10 Gy of radiation and anti-PD-L1 antibodyimmunotherapy did
not show significant effects on E0771 primary tumor growth when using an orthotopic
tumor model. The time required for the tumor volume to exceed an endpoint of 1500mm3
was not significantly affected by all of the treatment methods used in the current
study. The results from the IVIS and the India-ink Assay suggest that E0771 might
not a good model for lung metastasis. However, the treatment response and the E0771
model were affected by a number of technical problems that render the evaluation
inconclusive. Solutions to some of these technical problems have been provided, enabling
future reseachers to replicate and improve on this study and futher determine the
treatment response and the usefulness of the E0771 model. Regarding the zenascope
measurement, the changes in Hb-Sat(%) may be correlated with the blood vessel growth
within the primary tumors, while the changes in total Hb were almost negligible. Nevertheless,
several limitions when performing the zenascope measurement have been listed, including
the pigmentation and fur of the skin of mice, the motion of the mice and/or the operator’s
hands, as well as the uncertainties in placing the optical probe onto the tumor. Further
research is needed to uncover the promise of this combined therapy and to further
verify the correlation among the changes in Hb-Sat(%), the changes in total Hb levels,
and the tumor physiological characteristics.</p>
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