Camptothecin enhances the anti-tumor effect of low-dose apatinib combined with PD-1 inhibitor on hepatocellular carcinoma

Apatinib has been shown to apply to a variety of solid tumors, including advanced hepatocellular carcinoma. Preclinical and preliminary clinical results confirmed the synergistic antitumor effects of apatinib in combination with anti-programmed death-1 (PD-1) inhibitors. In this study, we investigated camptothecin (CPT) enhances the anti-tumor effect of low-dose apatinib combined with PD-1 inhibitor on hepatocellular carcinoma. CPT combined with a PD-1 inhibitor enhances the anti-tumor effects of low-dose apatinib in hepatocellular carcinoma which was evaluated in making use of the H22 mouse model (n = 32), which was divided into four groups. Immunohistochemical staining and western blotting were used to detect nuclear factor erythroid 2-related factor 2 (Nrf2) as well as sequestosome 1 (p62), vascular endothelial growth factor A (VEGFA), vascular endothelial growth factor receptor 2 (VEGFR2), PD-1, and programmed cell death ligand 1 (PD-L1). The results showed that the average size of the tumor of the combination group (Group D) was significantly less than that of the apatinib + PD-1 inhibitor group (Group C). The expression levels of Nrf2, p62, VEGFA, VEGFR2, PD-1, and PD-L1 in the apatinib + PD-1 inhibitor group(Group C) were lower than those in the control group (Group A) (P < 0.05). The expression levels of these genes in the apatinib + PD-1 inhibitor group (Group C) were significantly lower in the combination group (Group D) (P < 0.05). There was no obvious difference in body weight and liver and kidney functions between the four groups of mice. In conclusion, CPT improves the anti-tumor effect of low-dose apatinib combined with PD-1 inhibitor on hepatocellular carcinoma


H22 HCC model
Animal experiments were conducted in accordance with the ARRIVE guidelines (Approval No. S0007) approved by the Animal Protection and Utilization Committee of the First Hospital of Shandong First Medical University (Jinan).Confirm that all experiments are conducted in accordance with relevant guidelines and regulations.The mice were purchased by Beijing Viton Lihua Company.H22 tumors were provided by the laboratory of Qianfoshan Hospital.CPT, Aptinib and PD-1 inhibitor were purchased from Jinan Shengshi Biotechnology Co. H22 tumors were provided by the laboratory of Qianfoshan Hospital.
When the tumors reached the size of soybean grains, a caliper was used to measure the volume of the tumors, with tumor volume (mm 3 ) = π/6 × length × width 35 .Thirty-two out of 40 mice with similar tumor sizes were selected for enrollment, while the remaining 8 mice did not meet the criteria for tumor size.The criteria for tumor size around 7-8 mm.A total of 32 mice survived in good condition until the end of the experiment and were euthanized by sodium pentobarbital injection.Mice were euthanized by slow intraperitoneal injection of 2% sodium pentobarbital (150-200 mg/kg) until death.Finally, tumors were dissected after euthanasia and weighed after tumor removal to analyze tumor volume and body weight.Analyses were performed using GraphPad Prism software (version 8; GraphPad Software, Inc.).32 mice with similar tumor size were randomly divided into 4 groups (group A) (saline), CPT group (group B) (3 mg/kg CPT), apatinib + PD-1 inhibitor group (group C) (60 mg/kg apatinib + 10 mg/kg PD-1 inhibitor), and apatinib + PD-1 inhibitor combined with CPT group (group D) (60 mg/kg apatinib + 10 mg/kg PD-1 inhibitor + 3 mg/kg CPT) [36][37][38] .CPT was injected intraperitoneally every 3 days, and apatinib was administered daily by gavage, while PD-1 inhibitor was injected intraperitoneally every 3 days.A total of 40 mouse models were established, of which 32 were eligible for enrollment; the 8 animals not enrolled were euthanized by sodium pentobarbital injection.
All animal welfare was taken into account, including minimizing pain and suffering, the use of painkillers or anesthetics, or special housing conditions.The experimental duration of the mouse model was 30 d.After the completion of the experimental objectives, the animals were treated in a scientific and humane manner to minimize their panic and suffering, and euthanasia was performed gently and quickly.By observing respiration, cardiac arrest, pupil, nerve reflexes and other indicators, the death was comprehensively judged, and it was confirmed that the experimental animals had died.

Serum biochemistry
The health and behavior of H22 mice model were monitored every day after H22 cells were inoculated subcutaneously into the mice.Blood was collected via the retro-orbital sinus and was centrifuged at 4 °C, 1000×g for 5 min.Aspartate aminotransferase, alanine aminotransferase, blood urea nitrogen, creatinine and total bilirubin levels were measured using an automatic biochemical analyzer.

Statistical analysis
Analyses were performed using GraphPad Prism software (version 8; GraphPad Software, Inc.).Data are presented as the mean ± SD.Comparisons between groups were performed using one-way ANOVA with the post hoc test Tukey's multiple comparison test.P < 0.05 was considered as indicating a statistically significant difference (Supplementary File S1).

Ethics approval and consent to participate
All animal experiments were performed according to the ARRIVE guidelines approved (approval no.S0007) by the Animal Care and Use Committee of the First Hospital of Shandong First Medical University (Jinan, China).

CPT enhances low-dose apatinib sensitivity in HCC by inhibiting the Nrf2/p62 pathway
When apatinib, PD-1 inhibitor, and CPT were administered, Nrf2 protein in mouse H22 tumor tissues was reduced and tumor growth was also inhibited after inhibiting Nrf2 protein expression.The effects of the combination of apatinib and PD-1 inhibitor and the combination of CPT are shown (Fig. 1(1)).The pre-treatment tumor volumes were 30.958 ± 2.315 mm 3 , 31.087 ± 2.470 mm 3 , 31.154 ± 1.251 mm 3 , 31.416 ± 2.113 mm 3 (P ≥ 0.05) in groups A, B, C, and D, respectively (Fig. 1).Tumor volumes after treatment were 161.531 ± 24.616 mm 3  www.nature.com/scientificreports/groups C and D was statistically significant (P < 0.05) (Fig. 1(2,3)).This indicated that apatinib combined with PD-1 inhibitor inhibited tumor growth, and this inhibitory effect was even more pronounced after the combined administration of CPT.
To detect the effects of apatinib, PD-1 inhibitor and CPT on the regulation of Nrf2 and p62 in vivo, western blotting and IHC staining were performed on mouse H22 tumor tissues.Treatment down-regulated Nrf2 expression in group B (P < 0.05) and decreased Nrf2 expression in group C (P < 0.05) compared with group A. However, Nrf2 expression was significantly decreased in group D (P < 0.05) compared with groups C. IHC and western blotting yielded comparable results (Fig. 2(1-4)).In addition, p62 was also affected in a similar manner (Fig. 2(5-8)).Apatinib combined with PD-1 inhibitor had an inhibitory effect on Nrf2 and p62, and the inhibition was more pronounced after the combined administration of CPT, suggesting that CPT could enhance HCC sensitivity to apatinib.

Combination of CPT, apatinib, and PD-1 inhibitor inhibits angiogenesis
In the mouse H22 tumor model, the expression levels of VEGF A, VEGFR2 and p-VEGFR2 were lower in groups B and C than in group A (P < 0.05), whereas in group D, the expression levels of VEGFA and VEGFR2 were significantly lower than in groups A and C (P < 0.05) (Fig. 3(1-8)).Similarly, the downstream target of VEGFR2, c-Myc, showed similar changes (Fig. 3(9,10)).This indicated that apatinib combined with PD-1 inhibitor could inhibit tumor angiogenesis by inhibiting the expression of VEGFA and VEGFR2, and then c-Myc, and the inhibitory effect was even more evident with the combined administration of CPT.

Combination of CPT, apatinib and PD-1 inhibitor improves tumor microenvironment
In the mouse H22 tumor model, the expression levels of CD4 and CD8 were higher in both groups B and C than in group A (P < 0.05), while in group D, the expression levels of CD4 and CD8 were significantly higher than in groups A and C (P < 0.05).(Fig. 4(1-4)).This indicated that the combined treatment had a synergistic effect  www.nature.com/scientificreports/and enhanced the activity of T cells.TGF-β showed similar changes (Fig. 4(5,6)).The expression levels of CD69, IL-6 and IFN-γ were higher in both groups B and C than in group A (P < 0.05), while in group D, the expression levels of CD69, IL-6 and IFN-γwere significantly higher than in groups A and C (P < 0.05).(Fig. 5(1-6)).The expression levels of PD-1 and PD-L1 were lower in group B and group C than in group A (P < 0.05); while in group D, the expression levels of PD-1 and PD-L1 were significantly lower than in groups A and C (P < 0.05) (Fig. 6(1-6)).This indicated that the combination therapy could improve the tumor microenvironment and promote immune activation.

Safety and tolerability of the combination of CPT, apatinib, and PD-1 inhibitor
There was no difference in body weight between mice in the four treatment groups after treatment and tumor resection.The body weights of the mice were 20.25 ± 1.79 g (A), 18.75 ± 0.83 g (B), 18.88 ± 1.05 g (C) and 19.25 ± 1.30 g (D) (P ≥ 0.05) (Fig. 7(1)).Serum analysis of mice showed that blood urea nitrogen, creatinine, total bilirubin, alanine transferase, and aspartate transferase were at normal levels without any treatment affecting liver and kidney functions (Fig. 7(2-6)).

Discussion
PD-1 inhibitors in combination with anti-angiogenic drugs such as apatinib induce enhanced therapeutic effects and may become may be a promising strategy to extend the benefits of PD-1 inhibitor therapy to a wider group of hepatocellular carcinoma patients.However, there are many side effects associated with the combination of the two, and we expected to reduce the dosage of apatinib to make it more effective at a lower dose, so we applied CPT in combination to reduce the side effects and improve the anti-tumor effect.Our findings further suggest that CPT enhances the antitumor effects of low-dose apatinib combined with PD-1 inhibitors in HCC.
Tumor angiogenesis is essential for tumor growth and metastasis.VEGFA is the most important angiogenic factor in the vascular endothelial growth factor family, playing an important role in the occurrence and development of tumors.Studies have confirmed that high levels of VEGFA are related to poor prognosis in tumors such as in gastric cancer, ovarian cancer, HCC, non-small cell lung cancer, and endometrial cancer [39][40][41][42] .During tumor growth, high metabolism leads to a hypoxic microenvironment within the tumor, which activates growth factors and induces angiogenesis.Previous studies have shown that downregulation of Nrf2 reduces angiogenesis 43,44 , CPT also inhibited the vessel density 30 .Apatinib is a highly selective multiple TKI that very selectively blocks VEGFR2.It has been reported that apatinib effectively inhibits tumor proliferation and migration by blocking the VEGF axis 45 .Chen et al. 46 reported that the inhibitory effect of apatinib on tumorigenesis may be bound up with the downregulation of VEGF and VEGFR2 expression in HCC.In this study, the expression levels of VEGFA and VEGFR2 in the CPT group were lower than those in the control group, confirming that CPT could inhibit the VEGFA signaling pathway.
Because of the pronounced pro-angiogenic effects of VEGFA, high VEGFA expression exacerbates tumor vascular abnormalities, poor perfusion, and inadequate oxygen supply.Hypoxic eventually regulates TME into an immunosuppressive environment 47,48 .Therefore, the hypoxia, angiogenesis, and immunosuppressive tumor microenvironment induced by VEGFA overexpression is definitely detrimental to PD-1 inhibitor 21 .A recent study demonstrated that low-dose apatinib combined with anti-pd-1 inhibitors optimizes the tumor microenvironment by alleviating hypoxia and promoting CD8 (+) T-cell infiltration 37 , which was reinforced in our study by the combined application of CPT.IL-6 modulates PD-L1 in tumor cells as well as modulating inflammatory and immune responses 49,50 .We noted that apatinib inhibited IL-6-mediated upregulation of PD-L1 in tumor cells.Apatinib at low doses significantly alleviated tumor hypoxia, increased CD4+ and CD8+ cell infiltration, and decreased TGF-β levels at certain time points, suggesting that angiogenesis inhibitors have a true immunomodulatory effect 37 .Studies by Schmittnaegel et al. 49 and Elizabeth et al. 50provide evidence that antiangiogenic drugs specifically improve anti-PD-1/PD-L1 therapy when promoting an immunostimulatory tumor microenvironment and tumor vascular normalization in various tumor models.CD69 is the earliest cell surface marker for activated T cells, and we also found that apatinib promoted CD69 expression and IFN-γ secretion.Therefore, our results suggest that apatinib may partially restore the activation of T cells by targeting the VEGFR2/PD-L1 signaling pathway in tumor cells, thus exerting its tumor suppressive effect, which was likewise enhanced by the combined application of CPT.In this study, CPT was confirmed to enhance the antitumor effect of low-dose apatinib combined with PD-1 inhibitor in HCC.
CPT was proved as a potent Nrf2 inhibitor among multitudinous agents 34 .CPT has a proven safety profile and is used clinically for chemotherapy drug 51,52 .In our previous studies, CPT was shown to be effective in inhibiting ROS levels and Nrf2 expression 30,34 .Increasing evidence suggests that Nrf2 plays a important role in autophagy regulation by forming positive feedback with p62.It has been proved that insufficient autophagy leads to p62 accumulation, which further segregates Keap1, a negative regulator of Nrf2, leading to Nrf2 stabilization 53 .Apatinib induces cellular autophagy and apoptosis by promoting ROS generation and inhibiting Nrf2 and p62 expression 5 .This study confirms that the combination treatment can downregulate Nrf2 by inhibiting the Nrf2 axis, which is beneficial for inhibiting the growth of HCC as well as inducing autophagy and apoptosis in tumor cells.Notably, our work is limited to some extent by the ability of animal models to mimic human TME and the hypothesis-generating nature of the study, which suggests the need for further evaluation in more mouse experimental systems.
In conclusion, this study confirms that CPT enhances the antitumor effect of low-dose apatinib combined with PD-1 inhibitors in HCC.

Figure 1 .
Figure 1.The effects of the combination of apatinib and PD-1 inhibitor and the combination of CPT in H22 models (1).The tumor volumes before (2) and after (3) treatment in A, B, C and D groups (n = 8).*P < 0.05 and # P < 0.05.

Figure 7 .
Figure 7. Panels A, B, C, D, E, and F show the statistical analysis of weight, AST, ALT, BUN, Cr, and TBIL.