ICG-001 exerts stronger effect in increasing anti-tumor efficacy and enhancing survival of radiotherapy in immune-proficient than immune-deficient mice
To determine the treatment efficacy of ICG-001 in combination with radiotherapy, the Hepa1-6 tumor-bearing nude mice were treated with ICG-001, radiotherapy, or combination of radiotherapy and ICG-001 (Fig. 1a). we destinated the day when the treatments started as day 0. In vehicle group, ICG-001 group, and radiotherapy group, the designated tumor volume endpoint (1000 mm3) was reached at day 8, 10, and 14, respectively (Fig. 1b). While radiotherapy and ICG-001 combination group reached the endpoint on day 22. Compared with radiotherapy alone, combination treatment significantly inhibited tumor growth (Fig. 1b and c; mean tumor volume ± SEM at day 12: 946.00 ± 54.00 mm3 radiotherapy vs. 653.00 ± 35.01 mm3 radiotherapy plus ICG-001, P < 0.0001). In addition, combination therapy significantly prolonged survival compared with radiotherapy alone, which was shown by higher median survival time (Fig. 1d; 10 days radiotherapy vs. 17 days radiotherapy plus ICG-001, P < 0.0001)
Interestingly, in Hepa1-6 tumor-bearing C57BL/6 mice, we found that radiotherapy alone and combination treatment group did not reach the endpoint yet until day 40. In addition, the combination of ICG-001 with radiotherapy almost eliminated the xenografts (Fig. 1e and f, mean tumor volume ± SEM at day 40: 787.75 ± 186.88 mm3 radiotherapy vs. 32.27 ± 32.27 mm3 radiotherapy plus ICG-001, P = 0.0011). Furthermore, the median survival time of the mice treated with vehicle, ICG-001, and radiotherapy alone was 20, 30, and 40 days, respectively. Whereas the survival of the mice in combination group was 90% at the end of observation period (Fig. 1g). In addition, the xenografts from C57BL/6 mice showed higher complete response rate than those in nude mice after the combination therapy (90% C57BL/6 mice vs. 0% nude mice). Therefore, although the addition of ICG-001 to radiotherapy significantly inhibited tumor growth and prolonged the survival time of tumor bearing mice in both immune-proficient C57BL/6 mice than immune-deficient nude mice, the synergistic effect was dramatically stronger with the presence of normal immune condition.
Combination of ICG-001 and radiotherapy improves immune microenvironment in mice HCC xenografts
Previous results have shown that ICG-001 displayed more substantial therapeutic effect with radiotherapy in the context of immune, we hypothesized that ICG-001 could regulate tumor immune microenvironment (TME) in combination with radiotherapy in HCC. To confirm this, we analyzed the alterations of varied tumor-infiltrating lymphocytes (TILs) in xenografts from Hepa1-6-bearing C57BL/6 mice treated with ICG-001, radiotherapy, or the combination. Compared with vehicle, radiotherapy evaluated the ratio of TIL CD8+/CD3+ T cells (Fig. 2a; 39.31% ± 6.00% vehicle vs. 51.54% ± 5.57% radiotherapy, P = 0.025). Meanwhile, radiotherapy alone also increased the number of TIL CD8+ T cells, but the difference did not reach statistical significance (Fig. 2b; 42.85 ± 28.93 vehicle vs. 120.70 ± 71.77 radiotherapy, P = 0.6666). Moreover, the addition of ICG-001 further increased the percentage (Fig. 2a; 51.54% ± 5.57% radiotherapy vs. 67.62% ± 7.91% radiotherapy plus ICG-001, P = 0.0021) of TIL CD8+ T cells, and the number (Fig. 2b; 120.70 ± 71.77 radiotherapy vs. 325.30 ± 204.60 radiotherapy plus ICG-001, P = 0.0321).
Next, we explored the role of ICG-001 on effector function of TIL CD8+ and CD4+ T cells. After the stimulation of IFN-γ production by phorbol 12-myristate 13-acetate (PMA) and ionomycin, we found that the percentage of TIL CD8+ T cells that produced IFN-γ in combination treatment group was significantly higher than that in other treatment groups (Fig. 2c; 23.03% ± 7.79% radiotherapy plus ICG-001 vs. 6.95% ± 2.65% vehicle, P < 0.0001; vs. 14.80% ± 3.926% ICG-001, P = 0.0424; vs. 12.48% ± 3.87% radiotherapy, P = 0.0073). We additionally examined the percentage of TIL CD4+ T cells that produced IFN-γ. Similar with CD8+ T cells, compared with other treatment regimens, the combination of ICG-001 and radiotherapy significantly increased the percentage of TIL IFN-γ+ CD4+ T cells (Fig. 2d; 34.13% ± 14.93% radiotherapy plus ICG-001 vs. 6.76% ± 4.87% vehicle, P < 0.0001; vs. 13.08% ± 4.82% ICG-001, P = 0.0018; vs. 10.53% ± 3.92% radiotherapy, P = 0.0005).
Besides the positive effect, radiation also confers negative effect on TME. In our study, the upregulated number of TIL Tregs was observed in radiotherapy alone group (Fig. 2e; 4.12 ± 1.50 vehicle vs. 6.25 ± 1.49 radiotherapy, P = 0.0246). The addition of ICG-001 to radiotherapy reduced the infiltration of Tregs (Fig. 2e; 6.25 ± 1.49 radiotherapy vs. 4.66 ± 0.86 radiotherapy plus ICG-001, P = 0.1225), but the difference did not reach statistical significance. Meanwhile, compared with radiotherapy, combination treatment significantly increased the CD8+/Tregs ratio (Fig. 2f; 23.02% ± 12.49% radiotherapy vs. 54.84% ± 16.88% radiotherapy plus ICG-001, P = 0.0398).
We also investigated the status of innate immunity in xenografts from the mice with different treatments. No differences in the numbers of macrophage, dendritic cells, NK cells, and M2/macrophage ratio were observed between radiotherapy and combination treatment (Fig. 3a, b, c, and d, Supplementary Fig. S2). However, we found that adding ICG-001 to radiotherapy dramatically increased the ratio of M1/macrophage (Fig. 3e; 44.00% ± 6.99% radiotherapy vs. 57.55% ± 7.11% radiotherapy plus ICG-001, P = 0.0234).
In summary, the addition of ICG-001 to radiotherapy activated TME in mice HCC xenografts by increasing the frequency and function of cytotoxic TILs (CD8+ and CD4+ T cells), and decreasing the infiltration of suppressive TILs (Tregs).
ICG-001 increases radiation-induced DNA damage of HCC cells in vitro and in vivo
Previously we have shown that ICG-001 increased radiosensitivity in nude mice, which implied that other than immune-editing, ICG-001 also played a critical role in radio-sensitizing regulation. To confirm this, by performing clonogenic survival assays, we found that ICG-001 inhibited colony formation of both Hepa1-6 and HCC-LM3 cells (Fig. 4a and b). These findings indicated the role of ICG-001 in increasing instinct radiosensitivity of HCC cells. Since it is widely reported that radiation-induced DNA damage is closely associated with radiosensitivity. Thus, we investigated the effect of ICG-001 on radiation-induced double-strand breaks (DSBs) by detecting the accumulation nuclear foci of γ-H2AX by immunofluorescence staining, a specific marker of DSBs, at a serial timepoints after IR treatment. We found that there were significantly more cells with > 10 foci in combination treatment group than radiation alone group (Fig. 4c and e). In addition, compared with radiotherapy, the addition of ICG-001 caused higher protein level of γ-H2AX post IR (Fig. 4d and f). Consistent with in vitro results, we observed more cell death and higher level of γ-H2AX in mice treated with combination treatment than radiotherapy alone (Fig. 4g and h). Collectively, our results demonstrated that ICG-001 enhanced radiation-induced DNA damage and increased radiosensitivity of HCC cells in vitro and in vivo.
ICG-001 promotes the anti-tumor efficacy and immune-improvement effect of radiation via activation of cGAS/STING
Previous reports have demonstrated that radiation promoted T cell infiltration through producing double-strand DNA (dsDNA) and activating cGAS/STING signaling pathway. In this study, we showed that the addition of ICG-001 to radiotherapy increased radiation-induced DNA damage, improved TME of HCC and prolonged HCC-bearing mice survival. Next, we investigated whether the anti-tumor and immune-editing role of ICG-001 in combination with radiotherapy relies on cGAS/STING signaling pathway activation. By analyzing the protein levels of several key factors involved in cGAS/STING signaling pathway, including cGAS, p-STING, and p-TBK1, in Hepa1-6 or HCC-LM3 cells treated with different treatment regimens. We found that ICG-001 further enhanced radiation-induced cGAS/STING signaling pathway activation in HCC cells (Fig. 5a, Supplementary Fig. S3). Consistent with these findings in vitro, similar results were observed in xenografts derived from the mice treated with radiotherapy or combination treatment (Fig. 5b). All these data implied that ICG-001 might exert an anti-tumor effect with radiotherapy by boosting the radiation-induced activation of cGAS/STING pathway in HCC, which shed light on the possibility that blockage of cGAS/STING pathway could weaken the synergistic therapeutic efficacy of ICG-001 with radiotherapy. By challenging the tumor-bearing mice with STING inhibitor C-176, we found that C-176 impaired the anti-tumor effect of the combination treatment (Fig. 5c and d; day 20: 499.00 ± 158.98 mm3 combination therapy vs. 1246.90 ± 393.74 mm3 combination therapy plus C-176, P < 0.0001). Moreover, C-176 decreased the percentage (Fig. 5e and Supplementary Fig. S4a; combination therapy 56.83% ± 11.92% vs. combination therapy plus C-176 34.33% ± 6.16%, P = 0.0003) and number of TIL CD8+ T cells (Fig. 5f; combination therapy 272.10 ± 136.00 vs. combination therapy plus C-176 54.33 ± 37.89, P = 0.0002), and the IFN-γ production activity of CD8+ T cells (Fig. 5g and Supplementary Fig. S4b; combination therapy 19.89% ± 5.31% vs. combination therapy plus C-176 6.92% ± 3.02%, P = 0.0006). Taken all these together, our findings indicated that ICG-001 exerted the synergistic tumor-control and TME-activation effect with radiotherapy in a cGAS/STING dependent manner.
The addition of ICG-001 to radiotherapy prevented HCC recurrence
We analyzed the memory status of CD8+ T cells in mice spleens. Compared with radiotherapy, the addition of ICG-001 increased the percentage of TIL CD8+ effector memory T (TEM) cells (Fig 6a; radiotherapy 18.28% ± 1.52% vs. radiotherapy plus ICG-001 39.23% ± 6.03%, P < 0.0001). It is well demonstrated that the activation and memory status of CD8+ effector T cells is closely related with long-term tumor control, next we determined the effect of ICG-001 on tumor recurrence. We re-implanted new HCC tumors into the contralateral flank of the tumor bearing mice with complete course of varied treatments, and then monitored the new tumors in each group. Results revealed that, compared with radiotherapy alone, the addition of ICG-001 retarded the speed of tumor growth and reduced the secondary tumor volume, suggestive of the potent inhibitory role of ICG-001 with radiotherapy in secondary tumor growth (Fig. 6b and c). These data suggested that combination treatment improved immunologic memory and delayed recurrence of HCC by increasing TEM cells in tumors.