Radiation enhanced the local and distant anti-tumor efficacy in dual immune checkpoint blockade therapy in osteosarcoma

<div><p>Radiation therapy has been long utilized as localized cancer treatment. Recent studies have also demonstrated that it has a distant effect by the enhanced immunity, but it rarely occurs. The purpose of this study was to investigate whether X-ray irradiation combined with anti-PD-L1 and anti-CTLA-4 antibodies (P1C4) provides a higher probability of this distant effect as well as enhanced local antitumor efficacy for osteosarcoma. LM8 mouse osteosarcoma cells were inoculated into both legs of C3H mice assigned to one of four groups, namely no treatment (No Tx), P1C4, X-ray irradiation (RAD) to the leg of one side, and combination (COMB) groups. Survival and treatment-related immune molecular changes were analyzed. Administration of P1C4 produced a tumor growth delay on day 30 in 18% of the mice. In contrast, combination therapy produced the strongest tumor growth inhibition not only at the irradiated tumor but also at unirradiated tumor in 67% of the mice. Accordingly, lung metastasis in the COMB group was strongly reduced by 98%, with a significant survival benefit. Unirradiated tumor in mice in the COMB group significantly recruited CD8 + tumor-infiltrating lymphocytes with a moderate reduction of Treg, producing a significant increase in the CD8/Treg ratio. These results suggest that radiation enhances the efficacy of P1C4 treatment against distant metastasis as well as local control in osteosarcoma. Our data suggest that radiation therapy combined with dual checkpoint blockade may be a promising therapeutic option for osteosarcoma.</p></div

Application of a Strained Natural Rubber at High Temperatures

Robustness of the natural rubber (NR) crystal at high temperatures is important because vehicle tires are easily subjected to high temperatures due to friction. To understand the robustness of crystals in the strained NR, as its plausible application to high temperatures, changes in the following structural parameters as a function of temperature were examined in this study by conducting two-dimensional wide-angle X-ray diffraction measurements: crystal lattice constants (a, b, c, and β), unit cell (volume, thermal expansivity, and orientation factor), degree of crystallinity, and crystallite (size, volume, and number density in the strained NR specimen). As a result for a vulcanized NR specimen subjected to a constant strain of 6, thermal shrinkage of the c-axis length was found in the heating process, while both a- and b-axis lengths were found to increase with an increase in the temperature. Furthermore, the volume of the crystal lattice was found to increase with the temperature, while the orientation degree of the crystal lattice remained unchanged. The degree of crystallinity was found to gradually decrease with temperature from the beginning of heating. As for the crystallite, the size along the stacking direction of the (200), (120), and (201) planes decreased with temperature, while that of the (002) plane (i.e., along the c-axis direction) was found to increase. These results indicate that crystallites grow in the NR main chain direction, while they are subjected to melting in the other perpendicular directions upon heating. However, it was found that the crystallite volume and the number density of crystallites in the strained NR specimen continuously decreased with the increasing temperature. The thermo-reversibility of all the structural parameters was also examined experimentally

Effect of P1C4 and/or X-ray irradiation on overall survival by groups of the P1C4 (N = 6), RAD (N = 6), and COMB (N = 7).

<p>P-values were determined by the log-rank test with the adjustment by Holm method. Abbreviations: P1C4: Anti-PD-L1 and anti-CTLA-4 antibodies; COMB: Anti-PD-L1 and anti-CTLA-4 antibodies with X-ray irradiation; and RAD: X-ray irradiation.</p

Changes in the tumor-infiltrated lymphocytes after treatment.

<p>(a) Representative plots of one animal per group for CD8 expression 20 days after tumor inoculation. (b) Representative plots of one animal per group for Treg expression 20 days after tumor inoculation. (c) Quantitative data of the proportion of CD8+ TILs. (d) Quantitative data of the proportion of FoxP3 + cells in CD4 + TILs. (e) Quantitative data of CD8 to Treg ratio. For CD8 analysis, the number of mice was 7, 11, 9, and 5 in the No Tx, P1C4, COMB, and RAD groups, respectively. Some of these mice were analyzed for Treg expression and CD8 to Treg ratio (No Tx; N = 5, P1C4; N = 5, COMB; N = 3, RAD; N = 4). P-values were determined by the Student t-test with Bonferroni correction; *, P<0.05, **, P<0.01. Bars show the median value. Abbreviations: No Tx: No treatment; P1C4: Anti-PD-L1 and anti-CTLA-4 antibodies; COMB: Anti-PD-L1 and anti-CTLA-4 antibodies with X-ray irradiation; and RAD: X-ray irradiation.</p

In vitro induction of PD-L1 72 hours after X-ray irradiation.

<p>(a) PD-L1 gene expression change analyzed by quantitative real-time PCR after X-ray irradiation. (b) Flow cytometric analysis of PD-L1 induction after X-ray irradiation. (c) Quantitative analysis of PD-L1 expression by flow cytometry after X-ray irradiation. All experiments were performed in triplicate or more. Error bars show standard deviation. P-values were determined by Tukey’s honestly significant difference tests; *, P<0.05, **, P<0.01.</p

Effect of P1C4 and/or X-ray irradiation on tumor volume change in irradiated and unirradiated tumors.

<p>(a) Tumor volume change each day was normalized to volume on day 9 in the No Tx (N = 13), P1C4 (N = 11), X-ray irradiation (N = 9), and combination groups (N = 12). (b) Quantitative analysis of tumor volume change at day 21 and day 30. P-values were determined by Turkey’s honestly significant difference test. All data including the outlier were included in the statistical analysis. *, P<0.05. **, P<0.01. (c) Proportion of mice with partial response or complete response. The numbers in the pie chart indicate the number of mice. On day 30,2of 11 mice (18%) in the P1C4 alone group experienced a partial response whereas 8 of 12 mice (67%) in the combined therapy group had a partial response in the unirradiated tumor. On day 39, only 1 of 11 mice (9%) in the P1C4 group experienced complete response whereas 5 of 12 mice (42%) did so in the COMB group. <i>P</i>-values were determined by the Fisher’s exact test for comparison of the partial and complete response, and progressive disease between the P1C4 and COMB groups. Abbreviations: No Tx: No treatment; P1C4: Anti-PD-L1 and anti-CTLA-4 antibodies; COMB: Anti-PD-L1 and anti-CTLA-4 antibodies with X-ray irradiation; RAD: X-ray irradiation; IR-leg: Irradiated leg; UnIR-leg: Unirradiated leg, Vd: Volume on each day, Vint: Volume on the day of the initial treatment, CR: Complete response, PR: Partial response, and PD: Progressive disease.</p

Effect of P1C4 and/or X-ray irradiation on distant metastasis.

<p>(a) Representatives of liver, lung and kidney metastases in the No Tx, P1C4, X-ray irradiation, and combination groups, (b) Hematoxylin-eosin staining for the corresponding organs in (a) (magnification; x100). Asterisk marks show representative micrometastases. Quantitative analysis of the number of gross metastatic nodules in the No Tx (N = 8), P1C4 (N = 6), X-ray irradiation (N = 5), and combination groups (N = 5) is shown for the (c) liver, (d) lungs, and (e) kidneys. Bars show the mean value. P-values were determined by Tukey’s honestly significant difference tests; *, P<0.05, **, P<0.01. Abbreviations: No Tx: No treatment; P1C4: Anti-PD-L1 and ani-CTLA-4 antibodies; COMB: Anti-PD-L1 and anti-CTLA-4 antibodies with X-ray irradiation; and RAD: X-ray irradiation.</p