Apatinib, a selective vascular endothelial growth factor receptor 2-tyrosine kinase inhibitor, has demonstrated activity against a wide range of solid tumors, including advanced hepatocellular carcinoma (HCC). Preclinical and preliminary clinical results have confirmed the synergistic antitumor effects of apatinib in combination with anti-programmed death-1 (PD-1) blockade. However, the immunologic mechanism of this combination therapy remains unclear. Here, using a syngeneic HCC mouse model, we demonstrated that treatment with apatinib resulted in attenuation of tumor growth and increased tumor vessel normalization. Moreover, our results indicated that natural killer cells, but not CD4+ or CD8+ T cells mediated the therapeutic efficacy of apatinib in HCC mouse models. As expected, the combined administration of apatinib and anti-PD-1 antibody into tumor-bearing mice generated potent immune responses resulting in a remarkable reduction of tumor growth. Furthermore, increased interferon-γ and decreased tumor necrosis factor-α and interleukin-6 levels were observed, suggesting the potential benefits of combination therapy with PD-1 blockade and apatinib in HCC.
Liver cancer is the fourth leading cause of cancer mortality . Hepatocellular carcinoma (HCC) accounts for about 85–90% of all primary liver malignancies, and the common clinical risk factors include chronic infection with hepatitis B virus or hepatitis C virus, along with heavy alcohol intake, steatohepatitis, and diabetes . Despite multimodal therapy, the overall survival of patients with HCC has not improved significantly over the past two decades [1,2,3].
In China, most patients with HCC are diagnosed at advanced stages, and systemic therapy is the only treatment option for patients who have unresectable or advanced HCC . In 2018, lenvatinib, a multitargeted tyrosine kinase inhibitor (TKI), was approved by the Food and Drug Administration (FDA) for use in the treatment of HCC, whereas sorafenib remains an effective frontline treatment option since 2008 [5, 6]. However, during the last 10 years, most TKIs have failed to improve or parallel the efficacy of sorafenib as frontline treatment, or increase overall survival in trials of second-line treatment compared with placebo [7,8,9]. Therefore, there is an unmet need for effective systemic treatment options for patients with advanced HCC, particularly after failure of first-line therapy.
Recently, immunotherapy has revolutionized cancer treatment, enabling effective control of previously incurable and highly aggressive cancers. Antibodies against anti-programmed death-1 (PD-1) and its ligand, PD-L1, are widely being used for the treatment of malignancies, including HCC [10, 11]. Compared to TKIs, they are effective regardless of the response to prior therapies and viral status, and can elicit a durable adaptive immune response . Notably, nivolumab, an anti-PD-1 (αPD-1) antibody, received accelerated FDA approval as second-line treatment for advanced HCC after failure or intolerance to sorafenib, based on the results from two single-arm studies CheckMate 040 . However, majority of the patients remain refractory, highlighting the need to develop strategies to increase the efficacy of immunotherapy. The combination of targeted therapies with immune checkpoint inhibitors has been tested in early phase trials, such as sorafenib (NCT03211416, NCT01658878, and NCT02988440), lenvatinib (NCT03418922 and NCT03006926), and other TKIs in combination with immune checkpoint inhibitors .
Apatinib, a selective vascular endothelial growth factor receptor 2-TKI, has been approved as a third-line treatment for patients with metastatic gastric cancer , and has shown promising therapeutic effects against diverse tumor types, including HCC [15,16,17]. In addition, preliminary results of a phase Ib clinical trial of SHR-1210 (a clinically available αPD-1 antibody) in combination with apatinib have shown promising efficacy in patients with advanced HCC . Subsequently, a multicenter, phase II trial is currently underway to confirm these results (NCT03463876). However, the synergistic effect between apatinib and αPD-1 therapy in HCC has not been explored completely.
Studies have shown that antiangiogenic agents that target tumor blood vessels are capable of pruning immature vessels and normalizing the vasculature, thus facilitating the delivery of drugs and immune effector cells into the tumor immunologic microenvironment (TIME) . Previous clinical data support the notion that inhibiting vascular endothelial growth factor A (VEGFA) signaling may improve T-cell-mediated antitumor immunity [20, 21]. However, our results showed that natural killer (NK) cells, but not CD4+ or CD8+ T cells mediated the therapeutic efficacy of apatinib in HCC mouse models. Besides, apatinib not only efficiently inhibited tumor growth and angiogenesis in HCC, but also elicited NK cell activation. Furthermore, the combined administration of apatinib and αPD-1 antibody into tumor-bearing mice resulted in a remarkable increase in antitumor efficacy. Overall, our preclinical findings suggest the use of apatinib as a tumor-conditioning agent that may increase the efficacy of immunotherapy via effectively suppressing angiogenesis, while increasing NK cell-mediated antitumor immune activity in an HCC mouse model.
Materials and methods
Cell culture and reagents
The mouse HCC line Hepa1-6 was maintained in our laboratory and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS; Gibco, Waltham, MA, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin at 37 °C in a humidified atmosphere containing 5% CO2. The authenticity of the cell line was verified by short tandem repeat analysis (Cellcook Biotech Co., Ltd., Guangzhou, China). Prior to use, cells were confirmed to be mycoplasma-negative using the Mycoplasma Detection Kit (Invitrogen, Carlsbad, CA, USA). Apatinib (YN-968D1) was provided by Jiangsu Hengrui Medicine Co., Ltd. (Lianyungang, China), and diluted in 0.5% (w/v) carboxymethyl cellulose.
Animal tumor experiments
Five-to-six-week-old female C57BL/6 mice were purchased from Beijing HFK Bioscience Co., Ltd. (Beijing, China) and kept in an animal facility under pathogen-free conditions. The handing of mice and experimental procedures were performed in accordance with the guidelines for experimental animals approved by the Ethical Committee of Tianjin Medicine University.
To evaluate the effect of apatinib on primary tumor growth, 1 × 106 Hepa1-6 cells were injected subcutaneously into the right flanks of mice. On day 7 post injection, the mice were randomly divided into two or four groups (n = 6 or 7 mice per group), and orally administered carboxymethyl cellulose or apatinib (100 mg/kg) daily for 4 weeks, or intraperitoneally injected with αPD-1 antibody (200 μg/mouse) every 5 days (total of 5 times). Tumor volume and weight were measured [tumor volume = (length × width2)/2] after sacrifice of mice.
Immunohistochemistry (IHC) and immunofluorescence (IF) assays
Tumor specimens were fixed with 4% paraformaldehyde, dehydrated, paraffin-embedded, and sliced into 5 μm sections. IHC staining was performed according to the manufacturer’s protocol (Zhongshan Golden Bridge, Beijing, China). Antibodies were purchased from Abcam (Cambridge, London, UK) or Cell Signaling Technology (CST, Danvers, MA, USA) as follows: CD31 (Abcam, ab9498), alpha-smooth muscle actin (α-SMA; Abcam, ab32575), Ki-67 (Abcam, ab16667), PD-L1 (CST, #64988), and natural cytotoxicity triggering receptor 1 (NCR1; Abcam, ab199128).
For IF staining, the tissue slides were unmasked using EDTA solution and co-stained with mouse anti-CD31 and rabbit anti-α-SMA antibodies. The sections were then incubated sequentially with FITC-labeled anti-mouse IgG (bs-0296G; Biosynthesis Biotechnology, Beijing, China), followed by incubation with Fluor555-labeled anti-rabbit IgG (CST, #4413). Next, the tissue sections were stained with DAPI (BioLegend, San Diego, CA, USA) for nuclear staining, and visualized and imaged under a positive fluorescence microscope (Carl Zeiss, Oberkochen, Germany). FITC, Fluor555, and DAPI images were taken under the same field.
Enzyme-linked immunosorbent assay (ELISA)
Mouse VEGFA, interleukin (IL)-6, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and transforming growth factor (TGF)-β levels in the supernatants were detected using an ELISA kit, according to the manufacturer’s instructions (Dakewe, Beijing, China).
Isolation of leukocytes
Tumors were harvested and minced using a razor blade, followed by digestion with collagenase IV (0.2 mg/mL; Life Technologies, Carlsbad, CA, USA), dispase (2 mg/mL, Life Technologies), and DNase I (0.1 mg/mL, Life Technologies) in RPMI-1640 medium (without any supplements) for 30 min at 37 °C. The cell suspension was filtered using a cell strainer (70 μm) and washed in washing buffer (phosphate buffered saline containing 2 mM EDTA and 2% FBS).
After blockade of Fc receptors by incubation with CD16/32 Ab (clone93; eBioscience, San Diego, CA, USA) for 30 min, the leukocytes were stained with the following extracellular fluorescence antibodies for 30 min at 4 °C: CD45 [allophycocyanin (APC)/Fire™ 750, clone 30-F11], CD11b (APC, clone M1/70), Gr-1 (PerCP/Cy5.5, clone RB6-8C5), F4/80 (FITC, clone BM8), CD3e (PerCP, clone 145-2c11), NK1.1 [phycoerythrin (PE), clone PK136], CD4 (Alexa Fluor® 488, clone GK1.5), CD8a (APC, clone 53-6.7), CD69 (PE/Cy7, clone H1.2F3), NKG2D (FITC, clone C7), NKG2A (APC, clone 16A11), T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT; PE/Cy7, clone 1G9), PD-1 (PE/Cy7, clone 29 F.1A12), or isotype controls. For CD107a staining, leukocytes were incubated with 10 ng/mL brefeldin A (PeproTech, Rocky Hill, NJ, USA) and CD107a (PE/Cy7, clone1D4B) for 4 h at 37 °C in a 5% CO2 incubator, followed by staining for extracellular markers. All the above antibodies were purchased from BioLegend (San Diego, CA, USA). Data were acquired using a BD FACSAria II instrument (BD Biosciences, San Jose, CA, USA) and analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA).
Statistical data were analyzed using GraphPad Prism 6.0 software. All experiments were performed in triplicates. Data were presented as means ± standard deviation. Two-tailed Student’s t test, one-way single factorial analysis of variance (ANOVA), or two-way ANOVA were performed to determine statistical significance. The differences were considered significant for p values < 0.05, 0.01, and 0.001.
Apatinib decreases tumor growth in HCC mouse model
Since HCC is characterized as a hyper-vascular tumor, suppressing angiogenesis may exert strong antitumor effects . Here, we analyzed the in vivo antitumor activity of apatinib in an HCC Hepa1-6 mouse model (Fig. 1a). As shown in Fig. 1b, administration of apatinib (100 mg/kg/day, equivalent to the clinical dosage of 750 mg) significantly altered tumor growth. Both tumor weight and volume decreased compared with the control group (Fig. 1c). In addition, Ki-67 expression was found to be significantly reduced in apatinib-treated tumor specimens, as indicated by IHC staining (Fig. 1d). These results demonstrate the antitumor activity of apatinib in the HCC mouse model.
To further characterize the effects of apatinib, the release of angiogenic and inflammatory cytokines in the serum of HCC mice was analyzed. The serum level of VEGFA, a well-known pro-angiogenic factor produced by fibroblasts, inflammatory cells, and tumor cells , was significantly reduced in apatinib-treated mice (Fig. 1e). TNF-α and IL-6, important inflammatory response mediators produced by monocytes and macrophages, and TGF-β, an immune-suppressive cytokine synthesized mainly by Tregs (regulatory T cells) and tumor cells, have been shown to promote angiogenesis. However, IFN-γ, an important proinflammatory cytokine secreted by activated T cells and NK cells, has been reported to inhibit tumor angiogenesis directly and indirectly [24, 25]. Here, our results showed that the levels of TGF-β, IFN-γ, TNF-α, and IL-6 remained unaffected (Fig. 1f).
Apatinib impairs tumor angiogenesis and normalizes the remaining vasculature
Next, we analyzed the changes in tumor vascularization in the HCC mouse model. Results showed that apatinib decreased the relative CD31+ vascular area in the tumors of mouse analyzed at the termination end point (Fig. 2a). These findings indicate the sustained antiangiogenic activity of apatinib in HCC. Previous research has suggested that coverage of endothelial cells by pericytes may be favorable for tumor vessel normalization . Next, we then analyzed vascular maturation by α-SMA staining to visualize pericytes. Both IHC and IF analysis showed that the perivascular cell coverage of the tumor blood vessels (calculated as the ratio of α-SMA+ or α-SMA+ CD31+ cells in CD31+ population) was significantly higher in the apatinib-treated group compared to that in the control group (Fig. 2b, c). These results indicate that apatinib effectively suppresses angiogenesis while increasing pericyte coverage of the remaining blood vessels in an HCC mouse model.
Apatinib promotes intratumoral NK cell accumulation
It has been demonstrated that vascular normalization may facilitate the trafficking and function of immune cells, especially effector T cells in various tumors [27, 28]. Thus, we assessed the intratumoral immunological changes post apatinib treatment by flow cytometry. Interestingly, apatinib treatment significantly altered the proportion and number of NK cells, but not CD8+ and CD4+ T cells in the tumor (Fig. 3a). Consistently, increased NK cell infiltration was also observed, as indicated by IHC staining of NCR1 (or NKp46) (Fig. 3b). In addition, we analyzed the effects of apatinib on immunosuppressive cells, including tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs), which could potentially attenuate or inhibit NK cell-mediated antitumor immune response in TIME. We found that the percentages and number of both CD11b+F4/80+ TAMs and CD11b+Gr-1+ MDSCs showed no significant differences between the apatinib-treated and control groups (Fig. 3c). These findings suggest that apatinib stimulates NK cell infiltration in tumors.
Apatinib activates tumor-infiltrating NK cells
NK cells are essential antitumor effector cells, and dysfunction of NK cells contributes to the progression of HCC . In this study, treatment with apatinib increased the proportion of cells positive for CD69 (active marker) and CD107a (marker for degranulation), indicating that apatinib significantly increases NK cell cytotoxicity (Fig. 4a). The activity of NK cells is modulated by a range of germline-encoded inhibitory and activating receptors that recognize their respective ligand(s) on target cells or antigen-presenting cells, and is regulated by an integrated balance of activation and inhibitory signals . Here, in the apatinib-treated group, NK cell activation was observed, as indicated by higher levels of the activating receptor NKG2D and lower levels of the co-inhibitory receptor TIGHT (Fig. 4b). However, the levels of the inhibitory receptor NKG2A and immune checkpoint receptor PD-1 were not found to be significantly altered (Fig. 4b). Therefore, these data imply that apatinib reverses the decreased effector function of NK cells in a tumor microenvironment.
Apatinib improves the antitumor activity of PD-1 blockage in HCC mouse model
We next assessed the efficacy of αPD-1 treatment alone or in combination with apatinib in the HCC mouse model (Fig. 5a). As expected, treatment with apatinib or αPD-1 alone showed comparable efficacy of tumor inhibition, while a combination treatment of apatinib and αPD-1 showed a significant delay in tumor growth compared with the control group (Fig. 5b–d). These data were in line with the results of phase Ib trials of apatinib and SHR-1210 (αPD-1 antibody) combination therapy that demonstrated manageable toxicity and encouraging clinical activity in patients with advanced HCC .
Furthermore, consistent with previous findings, the VEGFA serum level was reduced in the apatinib-treated group, as well as in both αPD-1 blockade groups with or without apatinib (Fig. 6a). Besides, a similar decline in PD-L1 expression in the tumor tissues was observed, as indicated by IHC staining (Fig. 6b). However, the changes in intratumoral PD-L1 expression following antiangiogenic therapy or immunotherapy remain controversial [28, 31, 32]. In addition, no differences in TGF-β levels were observed among groups. The levels of IFN-γ increased, but those of TNF-α and IL-6 reduced in the αPD-1 and apatinib combination therapy group as compared to the apatinib-treated and control groups (Fig. 6c). These data indicate the potential benefits of combination therapy of PD-1 blockade and apatinib for the treatment of HCC.
Angiogenesis is increasingly used as an immune modulator with potential for combinatorial use with checkpoint blockade [27, 28, 31]. Here, we reported that treatment with apatinib increased intratumoral NK cell infiltration and activated NK cells, following tumor growth attenuation and tumor vessel normalization, and generated potent immune responses when used in combination with PD-1 blockade in the HCC mouse model. Our results, along with previous preliminary clinical findings [18, 33], underscore the potential benefits of combining PD-1 blockade with apatinib in HCC.
Like CD8+ T cells, NK cells exert antitumor effects and play a crucial role in tumor immune surveillance. They are functionally inhibited in the tumor microenvironment [29, 30]. Moreover, the decreased percentage and dysfunction of liver NK cells have been shown to contribute to the development and progression of HCC, suggesting the critical role of NK cells [29, 34]. In our HCC mouse model, apatinib treatment increased intratumoral NK cell proportion, and enhanced the expression of NK cell activation marker CD69 and cytotoxicity molecule CD107a, indicating that NK cells play a critical role in mediating the therapeutic effects of apatinib. In addition, the expression of the activating receptor NKG2D on NK cells was significantly upregulated after apatinib treatment, while that of the co-inhibitory receptor TIGIT was downregulated, suggesting that apatinib has the ability to reverse NK cell dysfunction in tumors.
Simultaneously, we also analyzed other immune components. Previous reports  have suggested that a low dose of apatinib (60 mg/kg) increases infiltration of CD8+ T cells and hinders the recruitment of TAM in mouse lung cancer models. In our study, there were no significant alterations in the levels of CD8+ T cells and TAMs after apatinib treatment (100 mg/kg), although a similarly high perivascular cell coverage was observed. However, except for the dosage, the observation time and tumor type were different between the two studies. Moreover, preliminary results from a phase Ib clinical trial suggested a recommended phase II dose of 250 mg/day for apatinib in combination with SHR-1210, which was one third of the recommended dose of 750 mg/day for phase III trials. Therefore, more number of preclinical and clinical studies to determine the optimal dose of antiangiogenic agents is needed to explore the role of immune modulation and help elicit stronger NK cell responses.
Since αPD-1/PD-L1-based immunotherapy has led to a remarkable breakthrough in cancer treatment, its limited efficacy has prompted preclinical and clinical studies to extend the benefits to a larger population by combining it with conventional treatments. Antiangiogenic drugs have the potential to enhance the efficacy of immunotherapy, which is based on the theory that they contribute to an immunostimulatory TIME and regulate tumor vessel normalization [31, 35]. Therefore, several early phase studies are increasingly being conducted to explore the safety and tolerability of antiangiogenic TKIs, such as sorafenib (NCT03211416, NCT01658878, and NCT02988440), lenvatinib (NCT03418922 and NCT03006926), and apatinib (NCT03463876) in patients with HCC [13, 18]. Our findings strongly indicate that apatinib has the potential to enhance the therapeutic efficacy of αPD-1. In addition, Hsu et al. revealed that PD-1/PD-L1 blockade elicited an antitumor NK cell response . Hence, it appears that coactivation of NK cells is required for mediating the synergistic antitumor effects of apatinib and αPD-1. However, further studies are needed for detailed mechanistic evidences.
Increased clinical use of checkpoint inhibitors results in immune-related adverse events (irAEs). Cytokines associated with T-cell activation and autoimmune disease usually contribute to irAEs . Thus, in the present study, we assessed the serum levels of IFN-γ, TNF-α, and IL-6. A slight increase in IFN-γ, and decrease in TNF-α and IL-6 level was observed in the αPD-1 and apatinib combination therapy group. Interestingly, emerging studies have suggested that TNF-α and IL-6 contribute to resistance to αPD-1/PD-L1 therapy [38, 39]. Hence, whether a decrease in the serum level of TNF-α or IL-6 contributes to the synergistic effect of antiangiogenic drugs combined with immunotherapy requires further investigation.
In summary, our findings support the notion that antiangiogenic agents enhance host antitumor immunity, and might be involved in mediating the therapeutic effects of certain immunotherapies. To the best of our knowledge, this is the first study to demonstrate that NK cells, but not CD8+ T cells play a critical role in mediating the therapeutic effects of apatinib. However, further investigation in future clinical studies is required to validate our results.
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This work was supported in part by grants from the National Natural Science Foundation of China (No. 81703786; to YY) and the Tianjin Science and Technology Committee (No. 18JCZDJC36700; to ZP).
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The authors declare that they have no conflict of interest.
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Yang, Y., Wang, C., Sun, H. et al. Apatinib prevents natural killer cell dysfunction to enhance the efficacy of anti-PD-1 immunotherapy in hepatocellular carcinoma. Cancer Gene Ther (2020). https://doi.org/10.1038/s41417-020-0186-7