Effect of Radiation and Immune Checkpoint Blockade (ICB) on Tumor Metastasis

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Date

2017

Authors

Lam, Sai Kit

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Palmer, Gregory M.
Dewhirst, Mark W.

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Abstract

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).

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.

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.

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.

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Lam, Sai Kit (2017). Effect of Radiation and Immune Checkpoint Blockade (ICB) on Tumor Metastasis. Master's thesis, Duke University. Retrieved from https://hdl.handle.net/10161/15254.

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